Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE I)E CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter 1e Bureau Canadien des
Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
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PROTEIN BINDING MINIATURE PROTEINS
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Patent Application No.
09/840,085 filed April 24, 2001, which claims the benefit of U.S. Provisional
Application No. 60/199,408 filed April 24, 2000; No. 60/240,566 filed ~ctober
16,
2000; and U.S. Provisional Application No. 60/265,099 filed January 30, 2001;
and No.
60/271,368 filed February 23, 2001. This application also claims priority to
U.S.
Provisional Patent Application No. 60/517,496 filed on November 4, 2003. The
teachings of these referenced applications are incorporated herein by
reference in their
entirety.
FUNDING
Work described herein was funded, in whole or in part, by National Institutes
of
Health, Grant Numbers 5-R01-GM59483 and 1-RO1-GM65453-01. The United States
government has certain rights in the invention.
FIELD OF THE INVENTION
The present invention relates to a polypeptide scaffold, such as an avian
pancreatic polypeptide, that is modified by substitution of at least one amino
acid
residue that is exposed on the alpha helix domain of the polypeptide when the
polypeptide is in a tertiary form. The invention also relates to phage display
libraries
for such scaffolds.
BACKGROUND OF THE INVENTION
Many proteins recognize nucleic acids, other proteins or macromolecular
assemblies using a partially exposed alpha helix. Within the context of a
native protein
fold, such alpha helices are usually stabilized by extensive tertiary
interactions with
residues that may be distant in primary sequence from both the alpha helix and
from
each other. With notable exceptions (Armstrong et al., (1993) J. Mol. Biol.
230, 284-
291), removal of these tertiary interactions destabilizes the alpha helix and
results in
molecules that neither fold nor function in macromolecular recognition (Zondlo
&
Schepartz, (1999) J. Am. Chem. Soc. 121, 6938-6939). The ability to
recapitulate or
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perhaps even improve on the recognition properties of an alpha helix within
the context
of a small molecule should find utility in the design of synthetic mimetics or
inhibitors
of protein function (Cunningham et al., (1997) Curr. Opin. Struct. Biol. 7,
457-462) or
new tools for proteomics research.
Two fundamentally different approaches have been taken to bestow alpha
helical structure on otherwise unstructured peptide sequences. One approach
makes
use of modified amino acids or surrogates that favor helix initiation (Kemp et
al.,
(1991) J. Org. Chem. 56, 6683-6697) or helix propagation (Andrews & Tabor,
(1999)
Tetrahedron 55, 11711-11743; Blackwell & Grubbs, (1998) Angew. Chem. Int. Ed.
Eng. 37, 3281-3284; Schafineister et al., (2000) J. Am. Chem. Soc. 122, 5891-
5892).
Perhaps the greatest success has been realized by joining the i and i+7
positions of a
peptide with a long-range disulfide bond to generate molecules whose helical
structure
was retained at higher temperatures (Jackson et al., (1991) J. Am. Chem. Soc.
113,
9391-9392). A second approach (Cunningham et al., (1997) Curr. Opin. Struct.
Biol. 7,
X457-462; Nygren, (1997) Curr. Opin. Struct. Biol. 7, 463-469), is to pare the
extensive
tertiary structure surrounding a given recognition sequence to generate the
smallest
possible molecule possessing function. This strategy has generated minimized
versions
of the Z domain of protein A (fifty-nine amino acids) and atrial natriuretic
peptide
(twenty-eight amino acids). The two minimized proteins, at thirty-three and
fifteen
amino acids, respectively, displayed high biological activity (Braisted &
Wells, (1996)
Proc. Natl. Acad. Sci., USA 93, 5688-5692; Li et al., (1995) Science 270, 1657-
1660).
Despite this success, it is difficult to envision a simple and general
application of this
truncation strategy in the large number of cases where the alpha helical
epitope is
stabilized by residues scattered throughout the primary sequence.
In light of this limitation, a more flexible approach to protein minimization
called protein grafting has been employed. Schematically, protein grafting
involves
removing residues required for molecular recognition from their native alpha
helical
context and grafting them on the scaffold provided by small yet stable
proteins.
Numerous researchers have engineered protein scaffolds to present binding
residues on
a relatively small peptide carrier. These scaffolds are small polypeptides
onto which
residues critical for binding to a selected taxget can be grafted. The grafted
residues are
arranged in particular positions such that the spatial arrangement of these
residues
mimics that which is found in the native protein. These scaffolding systems
are
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commonly referred to as miniproteins. A common feature is that the binding
residues
are known before the miniprotein is constructed.
Examples of these miniproteins include the thirty-seven amino acid protein
charybdotoxin (Vita et al., (1995) Proc. Natl. Acad. Sci. USA 92, 6404-6408;
Vita et
al., (1998) Biopolymers 47, 93-100) and the thirty-six amino acid protein,
avian
pancreatic peptide (Zondlo & Schepartz, (1999) Am. Chem. Soc. 121, 6938-6939).
Avian pancreatic polypeptide (aPP) is a polypeptide in which residues fourteen
through
thirty-two form an alpha helix stabilized by hydrophobic contacts with an N-
terminal
type II polyproline (PPII) helix formed by residues one through eight. Because
of its
small size and stability, aPP is an excellent scaffold for protein grafting of
alpha helical
recognition epitopes (Zondlo & Schepartz, (1999) J. Am. Chem. Soc. 121, 6938-
6939).
SUMMARY OF THE INVENTION
The invention encompasses an avian pancreatic polypeptide modified by
substitution of at least one amino acid residue; this residue is exposed on
the alpha
helix domain of the polypeptide when the polypeptide is in a tertiary form. In
some
embodiments, the modified polypeptide contains at least six substituted
residues, while
in other embodiments it contains eight substituted residues, while in another
embodiment it contains ten substituted residues, while in yet another
embodiment it
contains at least twelve substituted residues.
The substituted residues are selected from a site on a known protein through
which interaction with another molecule occurs. For example, one or more amino
acid
residues present in (of) a site on a known protein through which the known
protein
interacts (e.g., binds) with a binding partner replace one or more amino acid
residues of
the avian pancreatic polypeptide. Known proteins include, but are not limited
to,
GCN4, CEBP, Max, Myc, MyoD, double minute two, Bcl-2, protein kinase A, Jun
and
Fos. In a preferred embodiment, the site on the known protein is a binding
site. In
some embodiments the modified avian pancreatic polypeptide is capable of
inhibiting
the interaction between the known protein and another molecule while in other
embodiments it is capable of enhancing the interaction. In some embodiments,
the
binding site is a DNA binding site while in others it is a protein binding
site. Preferred
DNA binding sites include, but are not limited to the CRE half site, the CEBP
site, the
MyoD half site and the Q50 engrailed variant site.
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The invention also encompasses a phage-display library comprising a plurality
of recombinant phage that express any of the aforementioned modified avian
pancreatic
polypeptides of the invention. In a related embodiment, the invention
encompasses a
phage-display library comprising a plurality of recombinant phage that express
a
protein scaffold modified by substitution of at least one amino acid residue,
this residue
being exposed on the polypeptide when the polypeptide is in a tertiary form.
In some
embodiments, the protein scaffold of the phage-display library comprises the
avian
pancreatic polypeptide. The invention also encompasses an isolated phage
selected
from the phage library of the invention.
In specific embodiments, the invention is a modified avian pancreatic
polypeptide (aPP) comprising substitution of at least one amino acid residue,
said at
least one residue being exposed on the alpha helix domain of the polypeptide
when the
polypeptide is in a tertiary form, wherein the modified polypeptide binds to a
target
protein. In specific embodiments, at least six amino acid residues, at least
eight amino
acid residues, at lease ten amino acid residues or at least twelve amino acid
residues are
substituted. In certain embodiments, the site is a protein-binding site. The
at least one
substituted residues can be from any site of a known protein through which the
known
protein interacts with its binding partner. The target protein can be, for
example, a
binding partner of the known protein, which can be, for example, a Bcl2
protein, p53, a
protein kinase inhibitor (PI~I), or CREB. The binding partner can be, for
example,
selected from the group consisting of a Bcl2 protein, MDM2, protein kinase A
or CBP.
A variety of modified polypeptides can be produced, such as a modified
polypeptide
that inhibits interaction between the known protein and the binding partner; a
modified
polypeptide that binds to a deep 'groove of the target protein; a modified
polypeptide
ZS that binds to the groove of the target protein, wherein the groove is more
than 6 ~ at
deepest point; a modified polypeptide that binds to a shallow groove of the
target
protein; a modified polypeptide that binds to the shallow groove of the target
protein,
wherein the groove is less than 61~ at deepest point. The modified polypeptide
can
bind to the target protein with a Kd of less than 1 micromolar or to the
target protein
with high specificity.
In certain embodiments, the modified polypeptide comprises an amino acid
sequence selected from the sequence represented in Figures 1, 2, 6, 8 or Table
1.
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Further embodiments of the invention are as follows, with reference to the
appended claims:
19. A stabilized miniature protein comprising a miniature protein and a second
portion comprising a stabilizing domain, wherein the miniature protein is a
modified
avian pancreatic polypeptide (aPP) comprising substitution of at least one
amino acid
residue, said at least one residue being exposed on the alpha helix domain of
the
polypeptide when the polypeptide is in a tertiary form, wherein the modified
polypeptide binds to a target protein.
20. The stabilized miniature protein of claim 19, wherein the second portion
is
a polypeptide covalently fused to the miniature protein.
21. The stabilized miniature protein of claim 20, wherein the second portion
is
selected from the group consisting of serum 'albumin and an IgG Fc domain.
22. The stabilized miniature protein of claim 19, wherein the second portion
is
a non-amino acid moiety.
23. The stabilized miniature protein of claim 19, wherein said miniature
protein
includes one or more modified amino acid residues selected from the group
consisting
of a phosphorylated amino acid, a glycosylated amino acid, a PEGylated amino
acid, a
farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid,
an amino
acid conjugated to a lipid moiety, and an amino acid conjugated to an organic
derivatizing agent.
24. A pharmaceutical preparation comprising: a) a modified polypeptide of
claim 1; and b) a pharmaceutically acceptable carrier.
25. The pharmaceutical preparation of claim 24, wherein said preparation is
substantially pyrogen free.
26. A pharmaceutical preparation comprising: a) a stabilized miniature protein
of claim 19; and b) a pharmaceutically acceptable carrier.
27. The pharmaceutical preparation of claim 26, wherein said preparation is
substantially pyrogen free.
28. A phage-display library comprising a plurality of recombinant phage that
express the modified avian pancreatic polypeptide of claim 1.
29. A phage-display library comprising a plurality of recombinant phage that
express a protein scaffold modified by substitution of at least one amino acid
residue,
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said at least one residue being exposed on the polypeptide when the
polypeptide is in a
tertiary form, wherein the modified polypeptide binds to a target protein.
30. The phage-display library of claim 29, wherein said protein scaffold
comprises an avian pancreatic polypeptide (aPP)
31. A phage selected from the library of claim 29.
32. An isolated avian pancreatic polypeptide modified by substitution of at
least one amino acid residue, wherein the modified polypeptide comprises a
sequence
selected from the group consisting of
(a) an amino acid sequence selected from Figures 1, 2, 6, 8, and Table 1;
(b) a fragment of at least twelve (12) amino acids of any amino acid sequence
selected
from Figures 1, 2, 6, 8, and Table 1;
(c) an amino acid sequence selected from Figures 1, 2, 6, 8, and Table 1;
comprising
one or more conservative amino acid substitutions;
(d) an amino acid sequence selected from Figures 1, 2, 6, 8, and Table 1;
comprising
one or more naturally occurring amino acid sequence substitutions; and
(e) an amino acid sequence at least 95% identical to any amino acid sequence
selected
from Figures 1, 2, 6, 8, and Table 1.
33. An isolated nucleic acid encoding any one of the polypeptides in claim 32.
34. A recombinant polynucleotide comprising a promoter sequence operably
linked to a nucleic acid of claim 33.
35. A cell transformed with a recombinant polynucleotide of claim 34.
36. The cell of claim 35, wherein the cell is a mammalian cell.
37. The cell of claim 35, wherein the cell is a human cell.
38'. A method of making a miniature protein, comprising:
a) culturing a cell under conditions suitable for expression of the miniature
protein,
wherein said cell is transformed with a recombinant polynucleotide of claim
34; and
b) recovering the miniature protein so expressed.
39. A method of preparing a miniature protein that modulates the interaction
between a known protein and another molecule, comprising the steps of
(a) identifying at least one amino acid residue that contributes to the
binding between a
known protein and another molecule; and
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(b) modifying an avian pancreatic polypeptide by substitution of said at least
one amino
acid residue, such that it is exposed on the alpha helix domain of the
polypeptide when
the polypeptide is in a tertiary form.
40. A method of identifying a miniature protein that modulates the interaction
between a known protein and another molecule, comprising isolating at least
one
recombinant phage clone from the phage display library of claim 29 that
displays a
protein scaffold that modulates the association between a known protein and
another
molecule.
41. A method for treating a subject having a disorder associated with abnormal
cell growth and differentiation, comprising administering to the subject an
effective
amount of a miniature protein.
42. The method of claim 41, wherein the disorder is selected from the group
consisting of inflammation, allergy, autoimmune diseases, infectious diseases,
and
tumors.
43. The method of claim 41, wherein the miniature protein is selected from the
group consisting of
a) a modified polypeptide of claim 1;
b) a stabilized miniature protein of claim 19; and
c) an modified avian pancreatic polypeptide of claim 32.
44. A method of activating p53 function in a cell, comprising contacting the
cell with a miniature protein or a stabilized miniature protein.
45. The method of claim 44, wherein the miniature protein or stabilized
miniature protein comprises an amino acid sequence selected from Figure 2.
46. The method of claim 44, wherein the miniature protein or stabilized
miniature protein inhibits binding of p53 to MDM2.
47. The method of claim 44, wherein the cell is a mammalian cell.
48. The method of claim 44, wherein the cell is a cancer cell.
49. A method of inhibiting a protein kinase activity in a cell, comprising
contacting the cell with a miniature protein or a stabilized miniature
protein.
50. The method of claim 49, wherein the miniature protein or stabilized
miniature protein comprises an amino acid sequence selected from Figure 6.
51. The method of claim 49, wherein the miniature protein or stabilized
miniature protein binds to the protein kinase.
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52. The method of claim 49, wherein the protein kinase is PKA.
53. The method of claim 49, wherein the miniature protein or stabilized
miniature protein is conjugated with a protein kinase inhibitor (PKI).
54. The method of claim 49, wherein the cell is a mammalian cell.
55. The method of claim 49, wherein the cell is a cancer cell.
56. A method of activating CBP function in a cell, comprising contacting the
cell with a miniature protein or a stabilized miniature protein.
57. The method of claim 56, wherein the miniature protein or stabilized
miniature protein comprises an amino acid sequence selected from Figure 8 and
Table
1.
58. The method of claim 56, wherein the miniature protein or stabilized
miniature protein binds to CBP.
59. The method of claim 56, wherein the miniature protein or stabilized
miniature protein activates transcription via a CBP-dependent pathway.
60. The method of claim 56, wherein the cell is a mammalian cell.
61. The method of claim 56, wherein the cell is a cancer cell.
62. Use of a miniature protein or stabilized miniature protein for making a
medicament for the treatment of a disorder associated with abnormal cell
growth and
differentiation.
63. The method of claim 62, wherein the disorder is selected from the group
consisting of inflammation, allergy, autoimmune diseases, infectious diseases,
and
tumors.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color.
Copies of
this patent with color drawings) will be provided by the Patent and Trademark
Office
upon request and payment of the necessary fee.
F~i ure 1- Seven distinct sequences isolated from BAI~LIB phage library.
Dissociation constants for miniature protein binding to Bcl-2 are shown on the
right.
Fi~uures 2A-B - (A) Protein grafting as applied to the design of miniature
protein ligands for MDM2. (B) Sequence alignment of aPP and p53AD. Residues in
yellow and blue stabilize the aPP hydrophobic core; those in red contribute to
the
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binding of MDM2. Residues varied in Library #1 are in purple. Each I~ reported
represents the equilibrium dissociation constant of the peptide~GST-MDM2
complex
determined by fluorescence polarization analysis. GST-MDM2 was over-expressed
in
BL21 cells using clone G.
Fi _~,tre 3 - Fluorescence polarization analysis of the affinity of GST-MDM2
for
selected peptides and the affinity of pP53-OS for selected proteins. Plots
illustrate the
fraction of fluorescein-labeled p53AD, pP53-O5, pP53-03, pP53-04, pP53-O1,
pP53-02
bound as a function of GST-MDM2 concentration; and the fraction of fluorescein-
labeled pP53-OS bound (O) as a function of (0) protein kinase A, (~) Fos, (o)
carbonic
anhydrase, (o) calmodulin. Each point represents the average of at least three
trials.
Error bars represent the standard error. Kd values were calculated as
described in
Heyduk, et al., P~oc Natl Acad Sci U S A 1990, 87, 1744. Inset: Competition
between
pP53-OS and p53AD for GST-MDM2, as monitored by fluorescence polarization
analysis. Plot illustrates the fraction of p53AD-Flu (10 nM) bound to GST-MDM2
(400 nM) at equilibrium as a function of added pP53-OS (4.5 ~,M - 0.18 ~.M).
Ki was
calculated using the Cheng-Prusoff equation.
Fiyre 4 - Circular dichroism analysis of pP53-OS and p53AD secondary
structure. Spectra were acquired in 0.5 x PBS at 25 °C using a Aviv
Model 62DS
spectrometer. (a) Plots illustrate the CD spectra of pP53-OS at 2.75 ~,M (~)
and 6.75
~,M (O) and p53AD at 3 ~M (~). Each data set represents the average of 10
scans.
Spectra were background corrected but were not smoothed. (b) Temperature
dependence of the CD spectrum of pP53-O5. OMB is in units of deg~cm2~dmol-1.
Fi re 5 - Structures of (A) the PKA catalytic subunit bound to PKIS_24 (Zheng,
et al., Acta Cryst 1993, D49, 362-5) and (B) the natural product K252a and
K252a-~.
Fee 6 - Design of PKA inhibitors. Residues that contribute significantly to
PKA inhibition are in red; residues that contribute to aPP folding are in blue
(oc-helix)
or yellow (PPII helix).
Fee 7 - Affinity and inhibitory potency of PKA ligands. Fluorescence
polarization analysis of the equilibrium affinity of PKIFI° (black),
1F1" (blue) and 2Fn
(orange) for PKA in the presence (A) and absence (B) of 100 ~,M ATP.
Inhibition of
the phosphotransferase activity of PKA (red), PKB (black), PKCa (blue), PKG
(green),
and CamKII (pink) by (C) K252a; (D) PKI-K252a; and (E) 1-K252a.
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Fi re 8 - Protein grafting applied to the KIDP~KIX interaction. (A) Schematic
representation of the protein grafting process. In the KIDP~KIX complex, the
backbone
of CREB KID helix B is in blue, the hydrophobic residues of helix B important
for
CBP KIX binding are in red, the PKA recognition site is in green, and the Ser
phosphate moiety is in blue (phosphorous) and white (oxygen). In aPP, residues
from
the oc-helix that form part of the hydrophobic core are in blue and residues
from the
polyproline helix are in orange. In PPI~ID Library 1, the Coc atoms at
randomized
positions are in orange. (B) Library design. The amino acid sequence of helix
B of
CREB KID is aligned with the sequence of the oc-helix of aPP. The amino acid
sequence of PPKID Library 1 is below. Residues important for aPP folding are
in blue,
the PKA recognition site is in green, and hydrophobic residues of helix B
important for
binding CBP KIX are in red. Randomized residues are represented by X in
orange.
(C) Comparison of the oc-helix-binding surfaces of Bcl-XL (left) and CBP KIX
(right).
Bcl-XL contains a deep (~7 ~) hydrophobic cleft that recognizes the Bak BH3 a-
helix.
CBP KIX binds the CREB KID helix B in a shallow depression (< 51~ at the
deepest
point) on its surface.
Fi re 9 - HisKIX-binding affinity of PPKID and control peptides measured by
fluorescence polarization. Serial dilutions of HisKIX were incubated with 25-
50 nM of
fluorescein-labeled peptide (peptideFl°) for 30 min at 25 °C.
Each point represents an
average of three independent samples; the error bars denote standard error.
Observed
polarization values were converted to fraction of peptideFU bound using P,n;,l
and P",
values derived from the best fit of the polarization data to equation (1).
Curves shown
are the best fit of fraction of peptideFi° bound values to the
equilibrium binding
equation (2). Fraction of phosphorylated peptideFlu bound values are indicated
with
circular symbols, and fraction of unphosphorylated peptideFl" bound values are
indicated with triangular symbols. (A) KID-ABP, KID-BP, peptide CP and PPKIDP
1-
3. (B) KID-ABU, KID-BU, peptide CU and PPKIDU 1-3. (C) PPI~IDP 4-5, PPKIDU 4-
5 and PPKIDU7-8. (D) PPKID6U, PPKID6P and PPKID6 S18E.
Fi r~u a 10 - Competition between KID-ABP and PPKID4P (solid circle) or
PPKID6U (open circle) for binding to HisKIX measured by fluorescence
polarization.
Serial dilutions of KID-ABP were incubated with 1.5 ~,M or 3.0 ~,M HisKIX and
25 nM
fluorescein-labeled PPKID4P or PPKID6U (peptideFl") for 60 min at 25
°C,
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respectively. Each point represents an average of three independent samples;
the error
bars denote standard error. Observed polarization values were converted to
fraction of
peptideFlu bound using experimentally determined Pm;" and P",~ values
corresponding
to the polarization of samples containing 25 nM peptideFl° alone and
peptideFi° with 1.5
~.M or 3.0 ~,M HisKIX, respectively. Curves shown represent the best fit of
fraction of
peptideFl" bound values to equation (3). The close agreement between the Kd of
the
KID-ABP~HisKIX complex and the ICso values determined here provides evidence
that
the fluorescein tag appended to KID-ABP contributes neither positively nor
negatively
to the stability of the KID-ABP~HisKIX complex.
Figure 11- Affinity of PPKID4P (blue circle), PPKID6U (blue triangle) and
KID-ABP (red circle) for GST-KIXy6soA measured by fluorescence polarization.
Serial
dilutions of GST-I~IXY6soA were incubated with 25 nM of fluorescein-labeled
peptide
(peptidePl") for 30 min at 25 °C. Each point represents an average of
three independent
samples; the error bars denote standard error. Observed polarization values
were
converted to fraction of peptideFU bound using Pm;,l and Pl"~ values derived
from the
best fit of the polarization data to equation (1). Curves shown are the best
fit of
fraction of peptideFl" bound values to the equilibrium binding equation (2).
Figure 12 - Specificity of protein surface recognition by PPKID and control
peptides measured by fluorescence polarization. Binding reactions containing
serially
diluted target protein and 25-50 nM of fluorescein-labeled peptides
{peptideFl°) were
incubated for 30 min at 25 °C. Each point represents the average
polarization of two to
three independent samples; error bars denote standard error. Observed
polarization
values were converted to fraction of peptideFl° bound using P",;" and
P",~ values derived
from the best fit of the polarization data to equation (1). Curves shown are
the best fit
of fraction of peptideFl" bound values to the equilibrium binding equation
(2). (A) Plot
illustrating the polarization of fluorescently-labeled PPKID4P, PPKID6U,
peptide CP
and peptide CU molecules as a function of target protein (carbonic anhydrase
II or
HisKIX) concentration. Circular and triangular symbols indicate that HisKIX
was used
as the target protein; the symbols are colored as in Figure 9 with the
exception of the
points for peptide CU, which are in orange for clarity. Square symbols
indicate that
carbonic anhydrase was used as the target protein. (B) Plot illustrating the
polarization
of fluorescently-labeled PPKID4P, PPKID6U and peptide CP molecules as a
function of
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target protein (calmodulin or HisKIX) concentration. Circular and triangular
symbols
indicate that HisKIX was used as the target protein, and the symbols are
colored as in
Figure 9. Square symbols indicate that calmodulin was used as the target
protein.
Figures 13A-I - Binding isotherms illustrating the equilibrium affinities of
PPKID4P, PPKID6U and KID-ABP for CBP KIX variants as determined by
fluorescence polarization analysis at 25 °C. Each plot illustrates the
fraction of 25 nM
(A-C) KID-ABP, (D-F) PPKID4P, or (G-I) PPKID6U bound as a function of the
concentration of CBP KIX variant (M). Observed polarization values were
converted
to fraction of ligandFU bound using P,r,;~, and Pm~ values derived from the
best fit of the
polarization data to equation (1). Curves shown represent the best fit of the
data to
equation (2). Phosphorylated ligands are indicated with circles, whereas
unphosphorylated ligands are indicated by triangles. Each point represents an
average
of three independent trials; error bars denote standard error.
Figures 14A-B - Close-up view of (A) phosphoserine and (B) hydrophobic
contacts in the KID-ABP~CBP KIX complex. The backbones of KID-ABP and CBP
KIX are depicted as red and blue ribbons, respectively; the side chains of
Y658, K662,
L603, K606 and Y650 (from CBP KIX) and 5133, L138, L141 and A145 (from~KID-
ABP) are shown explicitly.
Fib - Close-up view of packing between residues on the a-helix and PPII
helix in the aPP hydrophobic core.
Figures 16A-B - Affinities of PPKID4P variants for CBP KIX as determined by
equilibrium fluorescence polarization analysis. Each point represents an
average of
three independent trials. Observed polarization values were converted to
fraction of
ligandFn bound using Pl~;" and P",~ values derived from the best fit of the
polarization
data to equation (1). Curves shown are the best fit of fraction of ligandFl"
bound values
to the equilibrium binding equation (2). A) Binding isotherms for PPKID4P
variants
L17A, F20A, L24A, Y27A and L28A. B) Binding isotherms for PPKID4P variants
P2A, P2Z, PSA, PSZ, P8A and P8Z. Z indicates the substitution of sarcosine in
place
of alanine.
Figures 17A-C - (a) Transcriptional activation mediated by Gal4 DBD fusions
of PPKID4, PPKID6 and KID-AB in HEK293 cells in the absence (b) or presence
(c)
of excess p300. The potency of each activation domain (fold activation) was
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determined by dividing the R values measured in cells transfected with a Ga4
DBD
fusion by the R value measured in cells transfected with the pALl control. The
R value
refers to the ratio of the activity of firefly and Rinella luciferase measured
using the
Dual-Luciferase~ Reporter Assay System (Promega). Bars and standard error
represent the results from at least 3 independent trials. Where indicated, 5
~.M
forskolin was added to the culture media 6 h prior to harvesting cells. When
indicated,
cells were also transfected with an expression vector encoding full-length
p300 under
control of the CMV promoter (25 ng).
Fi ure 18 - Transcriptional iWibition by PPKID4P peptide in mammalian cells.
For each peptide, 5 ng of the Gal4 DNA-binding domain fusion construct. For
each
peptide, HEI~93 cells were transfected with 5 ng of the Gal4 DBD fusion
construct
and indicated amount of PPI~ID4 vector (PPI~ID4 expressed without Gal4 DBD)
and
assayed for activation. Bars and standard error represent the results from at
least 3
independent trials. Firefly luciferase values were normalized to an internal
control
(luciferase values from promoterless Renilla luciferase vector) to correct for
transfection efficiency. Fold activation represents normalized luciferase
relative to
values for Gal4 DBD alone under the same. Where phosphorylation is indicated,
5 ~,M
forskolin was added to media 6 hours before harvesting cells.
DETAILED DESCRIPTION
Definitions
As used herein, the term "binding" refers to the specific association or other
specific interaction between two molecular species, such as, but not limited
to, protein-
DNA interactions and protein-protein interactions. The specific association
can be, for
example, between proteins and their DNA targets, receptors and their ligands,
enzymes
and their substrates. It is contemplated that such association is mediated
through
specific sites on each of the two interacting molecular species. Binding is
mediated by
structural and/or energetic components, the latter comprising the interaction
of
molecules with opposite charges.
As used herein, the term "binding site" refers to the reactive region or
domain
of a macromolecule that directly participate in its specific binding with
another
molecule. For example, when referring to the binding site on a protein or
nucleic acid,
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binding occurs as a result of the presence of specific amino acids or
nucleotide
sequence, respectively, that interact with the other molecule and,
collectively, are
referred to as a "binding site."
As used herein, the term "exposed on the alpha helix domain" means that an
amino acid substituted, for example, into the avian pancreatic polypeptide is
available
for association or interaction with another molecule and axe not otherwise
bound to or
associated with another amino acid residue on the avian pancreatic
polypeptide. This
term is used interchangeably with the term "solvent-exposed alpha helical
face"
throughout the specification.
As used herein, the terms "miniature protein" or "miniprotein" refers to a
relatively small protein containing at least a protein scaffold and one or
more additional
domains or regions that help to stabilize its tertiary structure. The term
"miniature
protein" includes any variants of the miniature protein (e.g., mutants,
fragments,
fusions, and peptidomimetic forms) that retain a useful activity. An exemplary
protein
scaffold is an avian pancreatic polypeptide (aPP). In certain specific
embodiments, a
miniature protein is referred to as "modified aPP" or "modified polypeptide."
As used herein, the term "modulate" refers to an alteration in the association
between two molecular species, for example, the effectiveness of a biological
agent to
interact with its target by altering the characteristics of the interaction in
a competitive
or non-competitive manner.
As used herein, the term "protein" refers to any of a group of complex organic
compounds which contain carbon, hydrogen, oxygen, nitrogen and usually
sulphur, the
characteristic element being nitrogen and which are widely distributed in
plants and
animals. Twenty' different amino acids are commonly found in proteins and each
protein has a unique, genetically defined amino acid sequence which determines
its
specific shape and function. The term "protein" is generally used herein
interchangeably with the terms peptide and polypeptide.
As used herein, the term "protein scaffold" refers to a region or domain of a
relatively small protein, such as a miniature protein, that has a conserved
tertiary
structural motif which can be modified to display one or more specific amino
acid
residues in a fixed conformation.
Miniature Proteins
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The present invention provides engineered miniature proteins that associate
with (i.e., or bind to) specific sequences of DNA or other proteins and also
provides
methods for designing and making these miniature proteins. These miniature
proteins
bind, for example, to DNA or other proteins with high affinity and
selectivity.
S Schematically, the invention involves a technique that the inventors have
designated as
protein grafting (see, e.g., Figures 2, 6, and 8). In one aspect, this
technique identifies
critical binding site residues from a globular protein that participate in
binding-type
association between that protein and its specific binding partners, then these
residues
are grafted onto, a small but stable protein scaffold. The preferred protein
scaffolds of
the invention comprise members of the pancreatic fold (PP fold) protein
family,
particularly the avian pancreatic polypeptide (aPP). Thus, in certain specific
embodiments, the miniature protein is referred to a modified polypeptide such
as a
modified aPP.
The PP fold protein scaffolds of the invention generally contain thirty-six
amino
acids andlare the smallest known globular protein. For example, an avian
pancreatic
polypeptide (aPP) sequence is designed as SEQ ID NO: 36. Despite their small
size,
PP fold proteins are stable and remain folded under physiological conditions.
The
preferred PP fold protein scaffolds of the invention consist of two anti-
parallel helices,
an N-terminal type II polyproline helix (PPII) between amino acid residues two
and
eight and an alpha-helix between residues 14 and 31 and/or 32. The stability
of the PP
fold protein scaffolds of the invention derives predominantly from
interactions between
hydrophobic residues on the interior face of the alpha-helix at positions 17,
20, 24, 27,
28, 30 & 31 and the residues on the two edges of the polyproline helix at
positions 2, 4,
5, 7 & 8. In general, the residues responsible for stabilizing it tertiary
structure are not
substituted in order to maintain the tertiary structure of the miniature
protein or are
compensated for using phage display.
In certain embodiments, two or more of the critical binding site residues of,
for
example, a selected globular protein are grafted onto the protein scaffold in
positions
which are not essential in maintaining tertiary structure, preferably on the
solvent-
exposed alpha helical face. In one preferred embodiment, six or more of such
binding
site residues are grafted onto the protein scaffold. In a more preferred
embodiment,
eight or more of such binding site residues are grafted onto the protein
scaffold. In an
even more preferred embodiment, ten or more of such binding site residues are
grafted
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onto the protein scaffold. In a most preferred embodiment, twelve or more of
such
binding site residues are grafted onto the protein scaffold. Preferred
positions for
grafting these binding site residues on the protein scaffold include, but are
not limited
to, positions on the solvent-exposed alpha-helical face of aPP. Substitutions
of binding
site residues may be made, although they are less preferred, for residues
involved in
stabilizing the tertiary structure of the miniature protein.
The skilled artisan will readily recognize that it is not necessary that
actual
substitution of the grafted residues occur on the protein scaffold. Rather it
is necessary
that a peptide be identified, through, for example, phage display, that
comprises a
polypeptide constituting a miniature protein having the association
characteristics of
the present invention. Such peptides may be produced using any conventional
means,
including, but not limited to synthetic and recombinant techniques.
Members of the PP fold family of protein scaffolds which are contemplated by
the present invention include, but are not limited to, avian pancreatic
polypeptide (aPP),
Neuropeptide Y, lower intestinal hormone polypeptide and pancreatic peptide.
In the
most preferred embodiment, the protein scaffold comprises the PP fold protein,
avian
pancreatic polypeptide (SEQ ID NO: 06} (see, e.g., Blundell et al., (1981)
Proc. Natl.
Acad. Sci. USA 78, 4175-4179; Tonan et al., (1990) Biochemistry 29, 4424-
4429).
aPP is a PP fold polypeptide characterized by a short (eight residue) amino-
terminal
type II polyproline helix linked through a type I beta turn to an eighteen
residue alpha-
helix. Because of its small size and stability, aPP is an excellent protein
scaffold for,
e.g., protein grafting of alpha-helical recognition epitopes.
DNA-binding Miniature Proteins
In another aspect, the present invention encompasses miniature proteins that
bind to specific DNA sequences and further encompasses methods for making and
using such miniature proteins. In some embodiments, these DNA sequences
comprise
sites for known proteins that bind to that specific DNA sequence (contemplated
known
proteins would be, e.g., a promotor or regulator). For example, in the design
of a
DNA-binding miniature protein, the amino acid residues of a known protein that
participate in binding or other association of the protein to that particular
DNA
sequence are identified.
In some embodiments of the present invention, the relevant binding residues
are
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identified using three-dimensional models of a protein or protein complex
based on
crystallographic studies while in other embodiments they are identified by
studies of
deletion or substitution mutants of the protein. The residues that participate
in binding
of the protein to the specific DNA sequence axe then grafted onto those
positions of the
miniature protein that are not necessary to maintain the tertiary structure of
the protein
scaffold to form the DNA-binding miniature protein. The identification of such
positions can readily be determined empirically by persons skilled in the art.
Other
embodiments of the present invention involve the screening of a library of
modified
miniproteins that contain peptide species capable of specific association or
binding to
that specific DNA (or, in other cases, protein) sequence or motif
Generally, it is contemplated that any potential binding site on a DNA
sequence
can be targeted using the DNA binding miniature proteins of the invention.
Preferred
embodiments include helical structures which bind to the DNA binding site. In
some
embodiments, the binding involves a basic region leucine zipper (bZIP)
structure
(Konig & Richmond, (1995) J. Mol. Biol. 254, 657-667) while in other
embodiments
the structure involves a basic-helix-loop-helix (bHLH) structure (Shimizu et
al., (1997)
EMBO J. 16, 4689-4697). In another embodiment, the binding involves a
structure like
those found in homeodomain proteins (Scott & Weimer, (1984) Proc. Natl. Acad.
Sci.
81, 4115-4119). Preferred bZIP structures include, but are not limited to,
those found
iri GCN4 and C/EBP -delta (Suckow et al., (1993) EMBO J. 12, 1193-1200) while
preferred bHLH structures include, but are not limited to, those found in Max
(Ferre-
D'Amare et al., (1993) Nature 363, 38-45), Myc and MyoD (Ma et al., (1994)
Cell 77,
451-459). Preferred homeodomain structures include, but are not limited to,
those
found in the Q50 engrailed variant protein (Kissinger et al., (1990) Cell 63,
579-590).
In one embodiment, the invention encompasses a DNA-binding miniature
protein that binds to the cAMP Response Element (CRE) half site promotor DNA
sequence (ATGAC) (SEQ ID NO: 65). Essential residues for binding are
identified
from the protein GCN4 which is a bZIP protein which binds to this sequence.
These
residues are identified by utilizing the three-dimensional structure of the
GCN4 protein
which bind to the hsCRE and grafting these residues onto the protein scaffold.
By
grafting various combinations of residues on the solvent-exposed alpha-helical
face or
domain of aPP which are essential to binding of GCN4 (SEQ ID NO: 7) to the CRE
half site (hsCRE), a series of polyproline helix-basic region (PPBRsR)
molecules
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containing most or all of the DNA-contact residues of GCN4 and most or all of
the
folding residues of aPP is generated. This procedure generated three positions
(Tyr27,
Leu28 and Va130) where essential DNA-contact and aPP-folding residues occupied
a
single position on the helix.
Examples of the DNA-binding miniature proteins which bind to hsCRE include,
but are not limited to, the amino acid sequences depicted in SEQ ID NO: 11
(PPBR2sR), 12 (PPBR4sR), 13 (G27) & 14 (PPBR40sR).
In another embodiment, protein grafting was used for the design of a miniature
protein whose DNA binding properties mimic those of the CCAAT/enhancer protein
C/EBP-delta. C/EBP-delta is a member of the C/EBP sub-family of bZIP
transcription
factors that includes C/EBP-alpha, C/EBP-beta, C/EBP-gamma, C/EBP-delta and
C/EBP-epsilon. Although C/EBP proteins are members of the bZIP superfamily,
they
differ from CGN4 at several residues within the DNA recognition helix. In
particular,
D/EBP-delta and GCN4 differ at two of six residues that contact bases or
sugars and
three of six residues that contact phosphates in all published structures of
GCN4 DNA
complexes. These changes, as well as the substitution of tyrosine or alanine
at position
fifteen, contribute to the preferred interaction of C/EBP proteins with the
C/EBP site
(ATTGCGCAAT) (SEQ ID NO: 67) over the CRE site (ATGACGTCAT) (SEQ ID
NO: 68) recognized by GCN4.
For the design of PPEBP (polyproline-enhancer binding protein) according to
the present invention, the first step in the grafting protocol is alignment of
the alpha-
helix of aPP (residues 14-36) with the alpha-helical region of the protein of
interest.
Alignment of the aPP alpha-helix with residues 187-221 (the DNA-binding basic
segment) of human C/EBP-delta identified three conflict positions (27, 28 & 30
according to the aPP numbering system) where DNA-contact residues within C/EBP-
delta and folding residues within aPP occupied the same position on the helix.
The
PPEBP 1 sR (SEQ ID NO: 47) miniature protein of the invention contains
arginine
residues derived from C/EBP-delta at positions 27, 28 & 30 to preserve binding
affinity
because high-affinity DNA recognition by PPEBP miniature proteins is enhanced
by
retention of DNA-contact residues at these positions despite the concomitant
loss in
folding energy. In addition, tyrosine, asparagine and valine residues are
substituted at
positions 15, 23 & 26, respectively to foster specific recognition of the
C/EBP half site
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ATTGC (hsCEBP). Finally an alanine residue is inserted at position 31 in place
of the
potentially core-disrupting and complex-destabilizing aspartate found in C/EBP-
delta
and in place of the helix destabilizing valine present at this position of
app.
Examples of the DNA-binding miniature proteins which bind to the C/EBP site
include, but are not limited to, the amino acid sequences depicted in SEQ ID
NO: 47
(PPEBP 1 SR), 48 (PPEBP2sR) and 49 (EBP 1 sR).
Methods of Producing Miniature Proteins Using Phag_e Display
In some embodiments, a miniature protein is produced and selected using a
phage display method (McCafferty et al., (1990) Nature 348, 552-554). In such
a
method, display of recombinant miniature proteins on the surface of viruses
which
infect bacteria (bacteriophage or phage) make it possible to produce soluble,
recombinant miniature proteins having a wide range of affinities and kinetic
characteristics. To display the miniature proteins on the surface of phage, a
synthetic
gene encoding the miniature protein is inserted into the gene encoding a phage
surface
protein (pIII) and the recombinant fusion protein is expressed on the phage
surface
(McCafferty et al., (1990) Nature 348, 552-554; Hoogenboom et al., (1991)
Nucleic
Acids Res. 19, 4133-4137). Variability is introduced into the phage display
library to
select for miniature proteins which not only maintain their tertiary, helical
structure but
which also display increased affinity for a preselected target because the
critical (or
contributing but not critical) binding residues are optimally positioned on
the helical
structure.
Since the recombinant proteins on the surface of the phage are functional,
phage
bearing miniature proteins that bind with high-affinity to a particular target
DNA or
protein can be separated from non-binding or lower affinity phage by antigen
affinity
chromatography. Mixtures of phage are allowed to bind to the affinity matrix,
non-
binding or lower affinity phage are removed by washing, and bound phage are
eluted
by treatment with acid or alkali. Depending on the affinity of the miniature
protein for
its target, enrichment factors of twenty-fold to a million-fold are obtained
by a single
round of affinity selection. By infecting bacteria with the eluted phage,
however, more
phage can be grown and subjected to another round of selection. In this way,
an
enrichment of a thousand-fold in one round becomes a million-fold in two
rounds of
selection. Thus, even when enrichments in each round are law (Marks et al.,
(1991) J.
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Mol. Biol, 222, 581-597), multiple rounds of affinity selection leads to the
isolation of
rare phage and the genetic material contained within which encodes the
sequence of the
domain or motif of the recombinant miniature protein that binds or otherwise
specifically associates with it binding target.
In various embodiments of the invention, the methods disclosed herein are used
to produce a phage expression library encoding miniature proteins capable of
binding
to a DNA or to a protein that has already been selected using the protein
grafting
procedure described above. In such embodiments, phage display can be used to
identify miniature proteins that display an even higher affinity for a
particular target
DNA or protein than that of the miniature proteins produced without the aid of
phage
display. In yet another embodiment, the invention encompasses a universal
phage
display library that can be designed to display a combinatorial set of
epitopes or
binding sequences to permit the recognition of nucleic acids, proteins or
small
molecules by a miniature protein without prior knowledge of the natural
epitope or
specific binding residues or motifs natively used for recognition and
association.
Various structural modifications also are contemplated for the present
invention
that, for example, include the addition of restriction enzyme recognition
sites into the
polynucleotide sequence encoding the miniature protein that enable genetic
manipulation of these gene sequences. Accordingly, the re-engineered miniature
proteins can be ligated, for example, into an M13-derived bacteriophage
cloning vector
that permits expression of a fusion protein on the phage surface. These
methods allow
for selecting phage clones encoding fusion proteins that bind a target ligand
and can be
completed in a rapid manner allowing for high-throughput screening of
miniature
proteins to identify the miniature protein with the highest affinity and
selectivity for a
particular target.
According to the methods of the invention, a library of phage displaying
modified miniature proteins is incubated with the immobilized target DNA or
proteins
to select phage clones encoding miniature proteins that specifically bind to
or otherwise
specifically associate with the immobilized DNA or protein. This procedure
involves
immobilizing a oligonucleotide or polypeptide sample on a solid substrate. The
bound
phage are then dissociated from the immobilized oligonucleotide or polypeptide
and
amplified by growth in bacterial host cells. Individual viral plaques, each
expressing a
different recombinant miniature protein, are expanded to produce amounts of
protein
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sufficient to perform a binding assay. The DNA encoding this recombinant
binding
protein can be subsequently modified for ligation into a eukaryotic protein
expression
vector. The modified miniature protein, adapted for expression in eukaryotic
cells, is
ligated into a eukaryotic protein expression vector.
Phage display methods that can be used to make the miniature proteins of the
present invention include those disclosed in Brinkman et al., (1995) J.
Immunol.
Methods 182, 41-50; Ames et al., (1995) J. Immunol. Methods 184:177-186;
I~ettleborough et al., (1994) Eur. J. Immunol. 24, 952-958; Persic et al.,
(1997) Gene
187, 9-18; Burton et al., (1994) Adv. Immunol. 57, 191-280; U.S. Patent Nos.
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;
5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743, 5,837,500 &
5,969,108.
Protein-Binding Miniature Proteins and Variants thereof
The invention encompasses miniature proteins that bind to other proteins and
methods for malting these miniature proteins. The binding of the miniature
proteins
modulates protein-protein and/or protein-ligand interactions. Thus, in some
embodiments, the binding blocks the association (or specific binding) of
ligands and
receptors. The ligand can be either another protein but also can be any other
type of
molecule such as a chemical substrate. In one embodiment of the present
invention,
making the protein-binding miniature protein of the invention involves
identifying the
amino acid residues which are essential to binding of the ligand protein to
its target
receptor protein. In some embodiments, these essential residues are identified
using
three-dimensional models of a protein or protein complex which binds to or
interacts
with another protein based on crystallographic studies while in other
embodiments they
are identified by studies of deletion or substitution mutants of the protein.
The residues
that participate in binding of the protein to are then grafted onto those
positions which
are not necessary to maintain the tertiary structure of the protein scaffold
to form the
protein-binding miniature protein.
The structure of any protein which binds to another protein can be used to
derive the protein-binding miniature proteins of the invention. Preferred
embodiments
include helical structures such as those involved in protein-protein
interactions between
Fos and Jun (I~ouzarides & Ziff, (1988) Nature 336, 646-651); Bcl-2 and Bak
(Sattler
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et al., (1997) Science 275, 983-986); CBP-KIX and CREB-KID (Radhakrishnan et
al.,
(1997) Cell 91, 741-752); p53 and MDM2 (Kussie et al., (1996) Science 274, 948-
953); and a protein kinase and a protein kinase inhibitor (PKI) (Glass et al.,
(1989) J
Biol Chem 264, 14579-84). In some embodiments, the binding involves coiled
coil
protein structures and/or leucine zippers.
In one embodiment of the invention, the methods disclosed herein are used to
produce a miniature protein that binds to the Bcl-2 or Bcl-XL proteins
(Sattler et al.,
(1997) Science 275, 983-986). In this method, the protein grafting procedure
described
herein was applied to the Bak-BH3 binding domain to design a miniature protein
capable of binding to Bcl-XL. In this procedure, the primary sequence of a
protein of
interest is aligned with residues in the alpha helix of aPP. All possible
alignments of
the primary sequence of positions 74-92 of Bak with aPP axe assessed in two
ways.
First, the number of conflicts in a primary sequence alignment between
residues
important for hydrophobic core formation or maintenance of aPP helix dipole,
and
residues in Bak important for binding Bcl-XL was considered. Alignments with a
large
number of conflicts are eliminated as they would force selection between
sequences
that were well folded or have high affinity, but make it difficult to isolate
a molecule
with both these properties.
Structural models of the aPP based peptides that are associated or complexed
with the BH3 domain of Bcl-XL in each of the alignments are evaluated for
unfavorable
interactions or steric clashes between the VanderWaals surface of Bcl-XL and
the
backbone of the aPP scaffold. Structural models with multiple unfavorable
interactions
or steric clashes are eliminated from further consideration.
An alignment is identified with only a single conflict where structural
modeling
suggested no steric clashes. A phage display expression library of chimeric
peptides
ultimately was based on this alignment. The resulting library of peptides was
displayed
on the surface of M13 phage and used in selection and isolation of miniature
proteins
that bind Bcl with high-affinity. Examples of bcl2-binding miniature proteins
include,
but are not limited to, those sequences having a carboxyl portion sequence as
depicted
in SEQ ID NO: 23 (410.0), 24 (4101), 25 (4099) or 26 (4102). The amino
terminal
portion of the miniature proteins is understood to derive from the amino
terminal
portion (e.g., residues 1-19) of the aPP (SEQ ID NO: 6).
In another embodiment of the invention, the methods of the invention are used
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to produce a miniature protein that binds to the human double minute two
(MDM2).
The alpha-helical segments of p53 and aPP were aligned to identify three
critical
MDM2 contact residues (e.g., positions 22, 26, and 29) on the exposed alpha-
helical
face of aPP without substituting any aPP residues important for folding.
Because many
p53 residues within the p53 activation domain that interacts with MDM2 display
phi
and psi angles outside the ideal alpha-helical range, this application of
protein grafting
introduced diversity at five positions along the alpha-helix and the highest
affinity
ligands were selected using phage display. Examples of miniature proteins
which bind
to MDM2 include, but are not limited to, the amino acid sequences depicted in
Figure 2
(e.g., pP53-O1, pP53-02, pP53-03, pP53-04 and pP53-OS).
In another embodiment of the invention, the methods of the invention are used
to produce a miniature protein that binds to protein kinase K (PKA). The alpha-
helical
segments of a PKI and aPP were aligned to identify critical contact residues
on the
exposed alpha-helical face of aPP without substituting any aPP residues
important for
folding. Examples of miniature proteins which bind to PKA include, but are not
limited to, the amino acid sequences depicted in Figure 6.
In yet another embodiment of the invention, the methods of the invention are
used to produce a miniature protein that binds to CBP (e.g, the KIX domain).
The
alpha-helical segments of a CREB (e.g., the KID domain) and aPP were aligned
to
identify critical contact residues on the exposed alpha-helical face of aPP
without
substituting any aPP residues important for folding. Examples of miniature
proteins
which bind to CBP include, but are not limited to, the amino acid sequences
depicted in
Figure 8 and Table 1.
In certain embodiments, miniature proteins include fragments, functional
variants, and modified forms that have similar or the same biological
activities of their
corresponding wild-type miniature proteins. To illustrate, miniature proteins
of the
invention bind to a target protein and modulate (e.g., activate or inhibit) a
function of
the target protein. In certain cases, target proteins of the miniature
proteins are known
to play a role in cell proliferation and differentiation. Therefore, miniature
proteins of
the invention can be used for treating or preventing disorders associated with
abnormal
cell proliferation and differentiation (e.g., inflammation, allergy,
autoimmune diseases,
infectious diseases, and tumors).
In certain embodiments, miniature proteins of the present invention further
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include conservative variants of the miniature proteins herein described. As
used
herein, a conservative variant refers to a miniature protein comprising
alterations in the
amino acid sequence that do not substantially and adversely affect the binding
or
association capacity of the protein. A substitution, insertion or deletion is
said to
adversely affect the miniature protein when the altered sequence prevents or
disrupts a
function or activity associated with the protein. For example, the overall
charge,
structure or hydrophobic-hydrophilic properties of the miniature protein can
be altered
without adversely affecting an activity. Accordingly, the amino acid sequence
can be
altered, for example to render the peptide more hydrophobic or hydrophilic,
without
adversely affecting the activities of the miniature protein.
In certain embodiments, these variants, though possessing a slightly different
amino acid sequence than those recited above, will still have the same or
similar
properties associated with the miniature proteins such as those depicted in
SEQ ID
NOs: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,
31, 33, 34,
35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,
63, 64, 70, 71,
and 72; and Figures 1, 2, 6, 8, and Table 1.
Ordinarily, the conservative substitution variants, will have an amino acid
sequence having at least ninety percent amino acid sequence identity with the
miniature
sequences such as those set forth in SEQ ID NOs: 8, 9, 10, 11, 12, 13, 17, 18,
19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50,
51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 and 72; and Figures l, 2, 6, 8,
and Table l,
more preferably at least ninety-five percent, even more preferably at least
ninety-eight
percent, and most preferably at least ninety-nine percent. Identity ar
homology with
respect to such sequences is defined herein as the percentage of amino acid
residues in
the candidate sequence that are identical with the known peptides, after
aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent
homology, and not considering any conservative substitutions as part of the
sequence
identity. N-terminal, C-terminal or internal extensions, deletions, or
insertions into the
peptide sequence shall not be construed as affecting homology.
Thus, the miniature proteins of the present invention include molecules
comprising the amino acid sequence of SEQ ID NOs: 8, 9, 10, 11, 12, 13, 17,
18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49,
50, 51, 52, 53,
54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70, 71 and 72; and Figures 1, 2,
6, 8, and
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Table 1; fragments thereof having a consecutive sequence of at least about 20,
25, 30,
35 or more amino acid residues of the miniature proteins of the invention;
amino acid
sequence variants of such sequences wherein at least one amino acid residue
has been
inserted N- or C-terminal to, or within, the disclosed sequence; amino acid
sequence
variants of the disclosed sequences, or their fragments as defined above, that
have been
substituted by another residue. Contemplated variants further include those
derivatives
wherein the protein has been covalently modified by substitution, chemical,
enzymatic,
or other appropriate means with a moiety other than a naturally occurring
amino acid
(for example, a detectable moiety such as an enzyme or radioisotope).
In certain embodiments, the miniature proteins of the present invention can be
chemically synthesized using techniques known in the art such as conventional
Merrifield solid phase f Moc or t-Boc chemistry. Alternatively, the miniature
proteins
can be produced (recombinantly or by chemical synthesis).
In certain embodiments, the present invention contemplates making functional
variants by modifying the structure of a miniature protein for such purposes
as
enhancing therapeutic efficacy, or stability (e.g., ex vivo shelf life and
resistance to
proteolytic degradation in vivo). Such modified miniature proteins when
designed to
retain at least one activity of the wildtype form of the miniature proteins,
are considered
functional equivalents of the wildtype miniature proteins.
In certain embodiments, the miniature proteins of the present invention
include
peptidomimetics. As used herein, the term "peptidomimetic" includes chemically
modified peptides and peptide-like molecules that contain non-naturally
occurring
amino acids, peptoids, and the like. Peptidomimetics provide various
advantages over
a peptide, including enhanced stability when administered to a subject.
Methods for
identifying a peptidomimetic are well known in the art and include the
screening of
databases that contain libraries of potential peptidomimetics. For example,
the
Cambridge Structural Database contains a collection of greater than 300,000
compounds that have known crystal structures (Allen et al., Acta Crystallogr.
Section
B, 35:2331 (1979)). Where no crystal structure of a target molecule is
available, a
structure can be generated using, for example, the program CONCORD (Rusinko et
al.,
J. Chem. Inf. Comput. Sci. 29:251 (1989)). Another database, the Available
Chemicals
Directory (Molecular Design Limited, Informations Systems; San Leandro
Calif.),
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contains about 100,000 compounds that are commercially available and also can
be
searched to identify potential peptidomimetics of the miniature proteins.
To illustrate, by employing scanning mutagenesis to map the amino acid
residues of a miniature protein which are involved in binding to another
protein,
peptidomimetic compounds can be generated which mimic those residues involved
in
binding. For instance, non-hydrolyzable peptide analogs of such residues can
be
generated using benzodiazepine (e.g., see Freidinger et al., in Peptides:
Chemistry and
Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988),
azepine
(e.g., see Huffinan et al., in Peptides: Chemistry and Biology, G.R. Marshall
ed.,
ESCOM Publisher: Leiden, Netherlands, 1988), substituted gamma lactam rings
(Garvey et al., in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM
Publisher: Leiden, Netherlands, 1988), keto-methylene pseudopeptides (Ewenson
et al.,
(1986) J. Med. Chem. 29:295; and Ewenson et al., in Peptides: Structure and
Function
(Proceedings of the 9th American Peptide Symposium) Pierce Chemical Co.
Rockland,
IL, 1985), b-turn dipeptide cores (Nagai et al., (1985) Tetrahedron Lett
26:647; and
Sato et al., (1986) J Chem Soc Perkin Trans 1:1231), and b-aminoalcohols
(Gordon et
al., (1985) Biochem Biophys Res Commun 126:419; and Dann et al., (1986)
Biochem
Biophys Res Commun 134:71 ).
In certain embodiments, the miniature proteins of the invention may further
comprise post-translational modifications in addition to any that are
naturally present in
the miniature proteins. Such modifications include, but are not limited to,
acetylation,
carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a
result,
the modified miniature proteins may contain non-amino acid elements, such as
polyethylene glycols, lipids, poly- or mono-saccharide, and phosphates.
Effects of such
non-amino acid elements on the functionality of a miniature protein may be
tested by
methods such as those described in the working examples.
In certain aspects, functional variants or modified forms of the miniature
proteins include fusion proteins having at least a portion of the miniature
proteins and
one or more fusion domains. Well known examples of such fusion domains
include,
but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase
(GST),
thioredoxin, protein A, protein G, an irmnunoglobulin heavy chain constant
region (Fc),
maltose binding protein (MBP), or human serum albumin. A fusion domain may be
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selected so as to confer a desired property. For example, some fusion domains
are
particularly useful for isolation of the fusion proteins by affinity
chromatography. For
the purpose of affinity purification, relevant matrices for affinity
chromatography, such
as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used.
Many of
such matrices are available in "kit" form, such as the Pharmacia GST
purification
system and the QIAexpress~ system (Qiagen) useful with (HIS6) fusion partners.
As
another example, a fusion domain may be selected so as to facilitate detection
of the
miniature proteins. Examples of such detection domains include the various
fluorescent proteins (e.g., GFP) as well as "epitope tags," which are usually
short
peptide sequences for which a specific antibody is available. Well known
epitope tags
for which specific monoclonal antibodies are readily available include FLAG,
influenza
virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains
have a
protease cleavage site, such as for Factor Xa or Thrombin, which allows the
relevant
protease to partially digest the fusion proteins and thereby liberate the
recombinant
proteins therefrom. The liberated proteins can then be isolated from the
fusion domain
by subsequent chromatographic separation. In certain preferred embodiments, a
miniature protein is fused with a domain that stabilizes the miniature protein
in vivo (a
"stabilizer" domain). By "stabilizing" is meant anything that increases serum
half life,
regardless of whether this is because of decreased destruction, decreased
clearance by
the kidney, or other pharmacokinetic effect. Fusions with the Fc portion of an
immunoglobulin are known to confer desirable pharmacokinetic properties on a
wide
range of proteins. Likewise, fusions to human serum albumin can confer
desirable
properties.
Nucleic Acid Molecules Encoding Miniature Proteins
The present invention further provides nucleic acid molecules that encode the
miniature proteins comprising any of the amino acid sequences of SEQ ID NOs:
8, 9,
10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 31, 33,
34, 35, 36, 37,
47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 70,
71, and 72; and
Figures l, 2, 6, 8, and Table 1, and the related miniature proteins herein
described,
preferably in isolated form. As used herein, "nucleic acid" includes cDNA and
mRNA,
as well as nucleic acids based on alternative backbones or including
alternative bases
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whether derived from natural sources or synthesized.
As used herein, a nucleic acid molecule is said to be "isolated" when the
nucleic
acid molecule is substantially separated from contaminant nucleic acid
encoding other
polypeptides from the source of nucleic acid.
The present invention further provides fragments of the encoding nucleic acid
molecule. As used herein, a "fragment of an encoding nucleic acid molecule"
refers to
a portion of the entire protein encoding sequence of the miniature protein.
The size of
the fragment will be determined by the intended use. For example, if the
fragment is
chosen so as to encode an active portion of the protein, the fragment will
need to be
large enough to encode the functional regions) of the protein. The appropriate
size and
extent of such fragments can be determined empirically by persons skilled in
the art.
Modifications to the primary structure itself by deletion, addition, or
alteration
of the amino acids incorporated into the protein sequence during translation
can be
made without destroying the activity of the miniature protein. Such
substitutions or
other alterations result in miniature proteins having an amino acid sequence
encoded by
a nucleic acid falling within the contemplated scope of the present invention.
The present invention further provides recombinant DNA molecules that
contain a coding sequence. As used herein, a recombinant DNA molecule is a DNA
molecule that has been subjected to molecular manipulation. Methods for
generating
recombinant DNA molecules are well known in the art, for example, see Sambrook
et
al., (1989) Molecular Cloning - A Laboratory Manual, Cold Spring Harbor
Laboratory
Press. In the preferred recombinant DNA molecules, a coding DNA sequence is
operably linked to expression control sequences and vector sequences.
The choice of vector and expression control sequences to which one of the
protein family encoding sequences of the present invention is operably linked
depends
directly, as is well known in the art, on the functional properties desired
(e.g., protein
expression, and the host cell to be transformed). A vector of the present
invention may
be at least capable of directing the replication or insertion into the host
chromosome,
and preferably also expression, of the structural gene included in the
recombinant DNA
molecule.
Expression control elements that are used for regulating the expression of an
operably linked miniature protein encoding sequence are known in the art and
include,
but are not limited to, inducible promoters, constitutive promoters, secretion
signals,
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and other regulatory elements. Preferably, the inducible promoter is readily
controlled,
such as being responsive to a nutrient in the host cell's medium.
In one embodiment, the vector containing a coding nucleic acid molecule will
include a prokaryotic replicon, i. e., a DNA sequence having the ability to
direct
autonomous replication and maintenance of the recombinant DNA molecule extra-
chromosomal in a prokaryotic host cell, such as a bacterial host cell,
transformed
therewith. Such replicons are well known in the art. In addition, vectors that
include a
prokaryotic replicon may also include a gene whose expression confers a
detectable
marker such as a drug resistance. Typical of bacterial drug resistance genes
are those
that confer resistance to ampicillin or tetracycline.
Vectors that include a prokaryotic replicon can further include a prokaryotic
or
bacteriophage promoter capable of directing the expression (transcription and
translation) of the coding gene sequences in a bacterial host cell, such as E.
coli. A
promoter is an expression control element formed by a DNA sequence that
permits
binding of RNA polymerase and transcription to occur. Promoter sequences
compatible with bacterial hosts are typically provided in plasmid vectors
containing
convenient restriction sites for insertion of a DNA segment of the present
invention.
Any suitable prokaryotic host can be used to express a recombinant DNA
molecule
encoding a protein of the invention.
Expression vectors compatible with eukaryotic cells, preferably those
compatible with vertebrate cells, can also be used to form a recombinant DNA
molecule that contains a coding sequence. Eukaryotic cell expression vectors
are well
known in the art and are available from several commercial sources. Typically,
such
vectors are provided containing convenient restriction sites for insertion of
the desired
DNA segment.
Eukaryotic cell expression vectors used to construct the recombinant DNA
molecules of the present invention may further include a selectable marker
that is
effective in a eukaryotic cell, preferably a drug resistance selection marker.
A
preferred drug resistance marker is the gene whose expression results in
neomycin
resistance, i.e., the neomycin phosphotransferase (neo) gene. Southern et al.,
(1982) J.
Mol. Anal. Genet. 1, 327-341. Alternatively, the selectable marker can be
present on a
separate plasmid, the two vectors introduced by co-transfection of the host
cell, and
transfectants selected by culturing in the appropriate drug for the selectable
marker.
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Transformed Host Cells
The present invention further provides host cells transformed with a nucleic
acid molecule that encodes a miniature protein of the present invention. The
host cell
can be either prokaryotic or eukaryotic. Eukaryotic cells useful for
expression of a
miniature protein of the invention are not limited, so long as the cell line
is compatible
with cell culture methods and compatible with the propagation of the
expression vector
and expression of the gene product.
Transformation of appropriate cell hosts with a recombinant DNA molecule
encoding a miniature protein of the present invention is accomplished by well
known
methods that typically depend on the type of vector used and host system
employed.
With regard to transformation of prokaryotic host cells, electroporation and
salt
treatment methods can be employed (see, for example, Sambrook et al., (1989)
Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory Press;
Cohen et al., (1972) Proc. Natl. Acad. Sci. USA 69, 2110-2114). With regard to
transformation of vertebrate cells with vectors containing recombinant DNA,
electroporation, cationic lipid or salt treatment methods can be employed
(see, for
example, Graham et al., (1973) Virology 52, 456-467; Wigler et al., (1979)
Proc. Natl.
Acad. Sci. USA 76, 1373-1376).
Successfully transformed cells (cells that contain a recombinant DNA molecule
of the present invention), can be identified by well known techniques
including the
selection for a selectable marker. For example, cells resulting from the
introduction of
a recombinant DNA of the present invention can be cloned to produce single
colonies.
Cells from those colonies can be harvested, lysed and their DNA content
examined for
the presence of the recombinant DNA using a method such as that described by
Southern, (1975) J. Mol. Biol. 98, 503-517 or the proteins produced from the
cell
assayed via an immunological method.
Production of Recombinant Miniature Proteins
The present invention further provides methods for producing a miniature
protein of the invention using nucleic acid molecules herein described. In
general
terms, the production of a recombinant form of a protein typically involves
the
following steps: a nucleic acid molecule is obtained that encodes a protein of
the
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invention, such as the nucleic acid molecule encoding any of the miniature
proteins
depicted in SEQ ID NO: 8, 9, 10, 11, 12, 13, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27,
28, 29, 31, 33, 34, 35, 36, 37, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61,
62, 63, 64, 70, 71, and 72; and Figures 1, 2, 6, 8, and Table 1. The nucleic
acid
molecule is then preferably placed in operable linkage with suitable control
sequences,
as described above, to form an expression unit containing the protein open
reading
frame. The expression unit is used to transform a suitable host and the
transformed
host is cultured under conditions that allow the production of the recombinant
miniature protein. Optionally the recombinant miniature protein is isolated
from the
medium or from the cells; recovery and purification of the protein may not be
necessary in some instances where some impurities may be tolerated.
Each of the foregoing steps can be done in a variety of ways. The construction
of expression vectors that are operable in a variety of hosts is accomplished
using
appropriate replicons and control sequences, as set forth above. The control
sequences,
expression vectors, and transformation methods are dependent on the type of
host cell
used to express the gene. Suitable restriction sites, if not normally
available, can be
added to the ends of the coding sequence so as to provide an excisable gene to
insert
into these vectors. A skilled artisan can readily adapt any host/expression
system
known in the art for use with the nucleic acid molecules of the invention to
produce a
recombinant miniature protein.
Methods to Identify Binding Partners
The present invention provides methods for use in isolating and identifying
binding partners of the miniature proteins of the invention. In some
embodiments, a
miniature protein of the invention is mixed with a potential binding partner
or an
extract or fraction of a cell under conditions that allow the association of
potential
binding partners with the protein of the invention. After mixing, peptides,
polypeptides, proteins or other molecules that have become associated with a
miniature
protein of the invention are separated from the mixture. The binding partner
bound to
the protein of the invention can then be removed and further analyzed. To
identify and
isolate a binding partner, the entire miniature protein can be used.
Alternatively, a
fragment of the miniature protein which contains the binding domain can be
used.
As used herein, a "cellular extract" refers to a preparation or fraction which
is
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made from a lysed or disrupted cell. A variety of methods can be used to
obtain an
extract of a cell. Cells can be disrupted using either physical or chemical
disruption
methods. Examples of physical disruption methods include, but are not limited
to,
sonication and mechanical shearing. Examples of chemical lysis methods
include, but
are not limited to, detergent lysis and enzyme lysis. A skilled artisan can
readily adapt
methods for preparing cellular extracts in order to obtain extracts for use in
the present
methods.
~nce an extract of a cell is prepared, the extract is mixed with a miniature
protein of the invention under conditions in which association of the
miniature protein
with the binding partner can occur. A variety of conditions can be used, the
most
preferred being conditions that closely resemble conditions found in the
cytoplasm of a
human cell. Features such as osmolarity, pH, temperature, and the
concentration of
cellular extract used, can be varied to optimize the association of the
protein with the
binding partner.
After mixing under appropriate conditions, the bound complex is separated
from the mixture. A variety of techniques can be utilized to separate the
mixture. For
example, antibodies specific to a protein of the invention can be used to
immunoprecipitate the binding partner complex. Alternatively, standard
chemical
separation techniques such as chromatography and density-sediment
centrifugation can
be used.
After removal of non-associated cellular constituents found in the extract,
the
binding partner can be dissociated from the complex using conventional
methods. For
example, dissociation can be accomplished by altering the salt concentration
or pH of
the mixture. . '
To aid in separating associated binding partner pairs from the mixed extract,
the
miniature protein of the invention can be immobilized on a solid support. For
example,
the miniature protein can be attached to a nitrocellulose matrix or.acrylic
beads.
Attachment of the miniature protein to a solid support aids in separating
peptide-
bindirig partner pairs from other constituents found in the extract. The
identified
binding partners can be either a single DNA molecule or protein or a complex
made up
of two or more proteins. Alternatively, binding partners may be identified
using the
Alkaline Phosphatase fusion assay according to the procedures of Flanagan ~
Vanderhaeghen, (1998) Annu. Rev. Neurosci. 21, 309-345 or Takahashi et al.,
(1999)
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Cell 99, 59-69; the Far-Western assay according to the procedures of Takayama
et al.,
(1997) Methods Mol. Biol. 69, 171-184 or Sauder et al., J. Gen. Virol. (1996)
77, 991-
996 or identified through the use of epitope tagged proteins or GST fusion
proteins.
Alternatively, the nucleic acid molecules encoding a miniature protein of the
invention can be used in a yeast two-hybrid system. The yeast two-hybrid
system has
been used to identify other protein partner pairs and can readily be adapted
to employ
the nucleic acid molecules herein described (see, e.g., Stratagene
Hybrizap° two-hybrid
system).
Screening, Diagnostic & Therapeutic Uses
The miniature proteins (including variants thereof) of the invention are
particularly useful for drug screening to identify agents capable of binding
to the same
binding site as the miniature proteins. The miniature proteins are also useful
for
diagnostic purposes to identify the presence and/or detect the levels of DNA
or protein
that binds to the miniature proteins of the invention. In one diagnostic
embodiment, the
miniature proteins of the invention are included in a kit used to detect the
presence of a
particular DNA or protein in a biological sample. The miniature proteins of
the
invention also have therapeutic uses in the treatment of disease associated
with the
presence of a particular DNA or protein. In one therapeutic embodiment, the
miniature
proteins can be used to bind to DNA to promote or inhibit transcription, while
in
another therapeutic embodiment, the miniature proteins bind to a protein,
resulting in
inhibition or stimulation of the protein.
As described above, miniature proteins bind to target proteins (MDM2, CBP,
PKA, a Bcl2 protein, and variants thereof) which are implicated in cell
proliferation
and differentiation. Thus, in certain embodiments, the present invention
provides
methods of treating cancer in an individual suffering from a disorder
associated with
abnormal cell proliferation and differentiation by administering to the
individual a
therapeutically effective amount of a miniature protein as described above.
Examples
of such disorders include, but are not limited to, inflammation, allergy,
autoimmune
diseases, infectious diseases, and tumors (cancers).
In other embodiments, the invention provides methods of preventing or
reducing the onset of a disorder associated with abnormal cell proliferation
and
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differentiation in an individual through administering to the individual an
effective
amount of a miniature protein. These methods are particularly aimed at
therapeutic and
prophylactic treatments of animals, and more particularly, humans. The term
"preventing" is art-recognized, and when used in relation to a condition, such
as cancer,
is well understood in the art, and includes administration of a composition
which
reduces the frequency of, or delays the onset of, symptoms of a medical
condition
(here, cancer) in a subject relative to a subject who does not receive the
composition.
Thus, prevention of cancer includes, for example, reducing the number of
detectable
cancerous growths in a population of patients receiving a prophylactic
treatment
relative to an untreated control population, and/or delaying the appearance of
detectable
cancerous growths in a treated population versus an untreated control
population, e.g.,
by a statistically and/or clinically significant amount. Prevention of an
infection
includes, for example, reducing the number of diagnoses of the infection in a
treated
population versus an untreated control population, and/or delaying the onset
of
symptoms of the infection in a treated population versus an untreated control
population. Prevention of pain includes, for example, reducing the magnitude
of, or
alternatively delaying, pain sensations experienced by subjects in a treated
population
versus an untreated control population.
In certain embodiments of such methods, one or more miniature proteins
thereof can be administered, together (simultaneously) or at different times
(sequentially). In addition, a miniature protein can be administered with
another type of
compounds for treating cancer (see below). The two types of compounds may be
administered simultaneously or sequentially.
A wide array of conventional compounds have been shown to have anti-tumor
activities. These compounds have been used as pharmaceutical agents in
chemotherapy
to shrink solid tumors, prevent metastases and further growth, or decrease the
number
of malignant cells. Although chemotherapy has been effective in treating
various types
of malignancies, many anti-tumor compounds induce undesirable side effects. In
many
cases, when two or more different treatments are combined, the treatments may
work
synergistically and allow reduction of dosage of each of the treatments,
thereby
reducing the detrimental side effects exerted by each compound at higher
dosages. In
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other instances, malignancies that are refractory to a treatment may respond
to a
combination therapy of two or more different treatments.
Therefore, the subject miniature protein may be conjointly administered with a
conventional anti-tumor compound. Conventional anti-tumor compounds include,
merely to illustrate: aminoglutethimide, amsacrine, anastrozole, asparaginase,
bcg,
bicalutamide, bleomycin, buserelin, busulfan, camptothecin, capecitabine,
carboplatin,
carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine,
cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,
daunorubicin,
dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine,
etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil,
fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea,
idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole,
leucovorin,
leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone,
megestrol,
melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone,
nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate,
pentostatin,
plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin,
suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa,
titanocene
dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine,
vindesine, and
vinorelbine.
In another related embodiment, the invention contemplates the practice of the
method in conjunction with other anti-tumor therapies such as radiation. As
used
herein, the term "radiation" is intended to include any treatment of a
neoplastic cell or
subject by photons, neutrons, electrons, or other type of ionizing radiation.
Such
radiations include, but are not limited to, X-ray, gamma-radiation, or heavy
ion
particles, such as alpha or beta particles. Additionally, the radiation may be
radioactive.
Administration and Pharmaceutical Formulations
Miniature proteins (including variants thereof) of the present invention can
be
administered in various forms, depending on the disorder to be treated and the
age,
condition, and body weight of the patient, as is well known in the art. For
example,
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where the miniature proteins are to be administered orally, they may be
formulated as
tablets, capsules, granules, powders, or syrups; or for parenteral
administration, they
may be formulated as injections (intravenous, intramuscular, or subcutaneous),
drop
infusion preparations, or suppositories. For application by the ophthalmic
mucous
membrane route, they may be formulated as eye drops or eye ointments. These
formulations can be prepared by conventional means, and, if desired, the
active
ingredient may be mixed with any conventional additive, such as an excipient,
a binder,
a disintegrating agent, a lubricant, a corrigent, a solubilizing agent, a
suspension aid, an
emulsifying agent, or a coating agent. Although the dosage will vary depending
on the
symptoms, age and body weight of the patient, the nature and severity of the
disorder to
be treated or prevented, the route of administration and the form of the drug,
in general,
a daily dosage of from 0.01 to 2000 mg of the compound is recommended for an
adult
human patient, and this may be administered in a single dose or in divided
doses.
The precise time of administration and/or amount of the agent that will yield
the
most effective results in terms of efficacy of treatment in a given patient
will depend
upon the activity, pharmacokinetics, and bioavailability of a particular
compound,
physiological condition of the patient (including age, sex, disease type and
stage,
general physical condition, responsiveness to a given dosage, and type of
medication),
route of administration, etc. However, the above guidelines can be used as the
basis for
fine-tuning the treatment, e.g., determining the optimum time and/or amount of
administration, which will require no more than routine experimentation
consisting of
monitoring the subject and adjusting the dosage and/or timing.
The phrase "pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such as a liquid
or solid
filler, diluent, excipient, solvent or encapsulating material, involved in
carrying or
transporting the subject chemical from one organ or portion of the body, to
another
organ or portion of the body. Each carrier must be "acceptable" in the sense
of being
compatible with the other ingredients of the formulation and not injurious to
the
patient.
Formulations useful in the methods of the present invention include those
suitable for oral, nasal, topical (including buccal and sublingual), rectal,
vaginal,
aerosol, and/or parenteral administration. The formulations may conveniently
be
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presented in unit dosage form and may be prepared by any methods well known in
the
art of pharmacy. The amount of active ingredient which can be combined with a
carrier
material to produce a single dosage form will vary depending upon the host
being
treated and the particular mode of administration. The amount of active
ingredient
which can be combined with a carrier material to produce a single dosage form
will
generally be that amount of the compound which produces a therapeutic effect.
Generally, out of one hundred per cent, this amount will range from about 1
per cent to
about ninety-nine percent of active ingredient, preferably from about 5 per
cent to
about 70 per cent, most preferably from about 10 per cent to about 30 per
cent.
Formulations suitable for oral administration may be in the form of capsules,
cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and
acacia or
tragacanth), powders, granules, or as a solution or a suspension in an aqueous
or.non-
aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as
an elixir or
syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or
sucrose and .
acacia) and/or as mouthwashes, and the like, each containing a predetermined
amount
of a therapeutic agent as an active ingredient. A compound may also be
admiustered as
a bolus, electuary or paste.
Liquid dosage forms for oral administration include pharmaceutically
acceptable emulsions, microemulsions, solutions, suspensions, syrups, and
elixirs. In
addition to the active ingredient, the liquid dosage forms may contain inert
diluents
commonly used in the art, such as, for example, water or other solvents,
solubilizing
agents, and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl
acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene
glycol, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame
oils), glycerol,
tetrahydrofuryl alcohol, polyethylene glycols, and fatty acid esters of
sorbitan, and
mixtures thereof.
Formulations which are suitable for vaginal administration also include
pessaries, tampons, creams, gels, pastes, foams, or spray formulations
containing such
carriers as are known in the art to be appropriate. Dosage forms for the
topical or
transdermal administration of a therapeutic agent include powders, sprays,
ointments,
pastes, creams, lotions, gels, solutions, patches, and inhalants. The active
component
may be mixed under sterile conditions with a pharmaceutically acceptable
carrier, and
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with any preservatives, buffers, or propellants which may be required.
The therapeutic agent can be alternatively administered by aerosol. This is
accomplished by preparing an aqueous aerosol, liposomal preparation, or solid
particles
containing the compound. A nonaqueous (e.g., fluorocarbon propellant)
suspension
could be used. Sonic nebulizers are preferred because they minimize exposing
the
agent to shear, which can result in degradation of the compound. Ordinarily,
an
aqueous aerosol is made by formulating an aqueous solution or suspension of
the agent
together with conventional pharmaceutically acceptable carriers and
stabilizers.
Transdermal patches have the added advantage of providing controlled delivery
of an therapeutic agent to the body. Such dosage forms can be made by
dissolving or
dispersing the agent in the proper medium. Absorption enhancers can also be
used to
increase the flux of the therapeutic agent across the skin. The rate of such
flux can be
controlled by either providing a rate controlling membrane or dispersing the
peptidomimetic in a polymer matrix or gel.
Pharmaceutical compositions of this invention suitable for parenteral
administration comprise one or more miniature proteins in combination with one
or
more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous
solutions,
dispersions, suspensions or emulsions, or sterile powders which may be
reconstituted
into sterile injectable solutions or dispersions just prior to use, which may
contain
antioxidants, buffers, bacteriostats, solutes which render the formulation
isotonic with
the blood of the intended recipient or suspending or thickening agents.
In some cases, in order to prolong the effect of a drug, it is desirable to
slow the
absorption of the drug from subcutaneous or intramuscular injection. This may
be
accomplished by the use of a liquid suspension of crystalline or amorphous
material
having poor water solubility. The rate of absorption of the drug then depends
upon its
rate of dissolution which, in turn, may depend upon crystal size and
crystalline form.
Alternatively, delayed absorption of a parenterally administered drug form is
accomplished by dissolving or suspending the drug in an oil vehicle.
These miniature proteins may be administered to humans and other animals for
therapy by any suitable route of administration, including orally, nasally, as
by, for
example, a spray, rectally, intravaginally, parenterally, intracisternally,
and topically, as
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by powders, ointments or drops, including buccally and sublingually.
EXAMPLES
Without further description, it is believed that a person of ordinary skill in
the
art can, using the preceding description and the following illustrative
examples, make
and utilize the compounds of the present invention and practice the claimed
methods.
The following working examples therefore, specifically point out preferred
embodiments of the present invention, and are not to be construed as limiting
in any
way the remainder of the disclosure.
Example 1 - Synthesis of DNA-binding miniature proteins
Polypeptides constituting miniature proteins were prepared using solid phase
methodology and contain a carboxy-terminal amide and a free amino terminus
unless
otherwise indicated. High performance liquid chromatography (HPLC) was
performed
on either a Waters 600E Multisolvent Delivery System with a Waters 490E ' .
multiwavelength detector or a Rainin Dynamax SD-200 Solvent Delivery System
with
a Rainin Dynamax PDA-2 Diode Array Detector.
Solid phase peptide synthesis was performed on a Perseptive BioSearch 9600
peptide synthesizer. Standard research grade argon (Connecticut AirGas) was
passed
through an OxyClear oxygen scrubber before introduction to the synthesizer.
HATU
(O-(7-benzotrizol-1-yl)-1,1,3,3,-tetramethyl uronium hexafluorophosphate) was
used as
the activating reagent without addition of supplemental benzotrizole.
Dimethylformamide, piperidine and methylene chloride (Baker) were fresh and
stored
under nitrogen. Anhydrous dimethylformamide was mixed with
diisopropylethylamine
(DIPEA, redistilled 0.46 M) to prepare the base activator solution. 9-
Fluorenylmethoxycarbonyl (F-moc)-protected amino acids utilized the following
side
chain protecting groups: O-t-butyl (Asp, Glu); t-butyl (Tyr, Thr, Ser);
2,2,4,6,7-
pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) (Arg); t-butoxycarbonyl (Lys);
and
triphenylmethyl (Cys, His, Asn, Gln). Synthesis was performed on a 0.10 mmol
scale
using PAL (peptide amide linker) resin (Fmoc-NH2-CH2-(di-m-methoxy,p-O-
(CHZ)4C(O)-polystyrene) which resulted in an amidated carboxy-terminus. Fmoc-
amino acid and HATU were used in four-fold excess (0.4 mmol per coupling).
After
the final coupling was completed, the Fmoc-protecting group was removed and
the
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resin was washed for the last time. The resin was dried and stored in a
desicator until
cleavage and deprotection were initiated.
Reverse phase HPLC was performed using eluents composed of mixtures of
Buffer A (98% HPLC water, 2% acetonitrile, 0.05% trifluoroacetic acid) and
Buffer B
(20% HPLC water, 80% acetonitrile, 0.06% trifluoroacetic acid). All HPLC
solvents
were filtered through a 0.2 micron filter prior to use. Solvents and chemicals
for
peptide synthesis were obtained from Aldrich and Perceptive Biosearch unless
stated
otherwise. Peptides were lyophilized using a Savant SC100 Speed Vacuum
instrument.
Denaturing sodium dodecyl sulfate-polyacryalmide gel electrophoresis (SDS-
PAGE)
analysis was performed with a Pharmacia PhastGel system using High Density
gels
(20% acrylamide soaked in glycerol). Amino acid analysis was assayed on a
Beckman
Analyzer.
For deprotection and purification of PPEBP 1 sH, PAL resin (15 mg) containing
protected PPEBP 1 sH was allowed to react for five hours at room temperature
in a
deprotection cocktail (84% trifluoroacetic acid, 4% phenol, 4% ethanedithiol,
4%
thioanisole and 4% water). The solvent was removed by blowing a stream of
nitrogen
over the solution until the volume reached approximately 0.25 ml. Diethylether
(1 ml)
and dithiothreitol (20 mg) were added to precipitate the peptide and stabilize
the
cysteine. The supernatant was removed after centrifugation and the precipitate
dried.
The crude peptide was dissolved in 1 ml phosphate-buffered saline (pH 7.5)
with added
dithiothreitol (5 mg) and filtered with a 0.2 micron filter. The peptide was
purified by
reverse phase HPLC (Vydac semipreparative 300 ~ C18, 5 microns, 10.0 x 250 mm)
using a 120 minute linear gradient of 100 - 30% Buffer A in Buffer B. The
peptide
eluted at 49.3 minutes using a flow rate of 4 ml/min and was analyzed by
electrospray
ionization mass spectrometry. The predicted and observed masses were 4729.4
and
4730.0, respectively.
For preparation of PPEBP 1 sR, 0.080 mg of PPEBP 1 sH was dissolved in 0.50 ml
of 2 mg/ml (15 mM) 2-bromoacetasnide in 20 mM sodium phosphate buffer (pH
7.5).
The reaction was allowed to proceed for thirty minutes at room temperature.
The
peptide was purified by reverse phase HPLC (Rainin analytical 100 ~ C18, 5
microns,
4.6 x 250 mm) using a forty minute linear gradient of 100 - 30% Buffer A in
Buffer B.
The peptide eluted at 23.3 minutes using a flow rate of 1 ml/min and was
characterized
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by electrospray ionization mass spectrometry and amino acid analysis. AAA
expected:
Alas AsxS CmCysl Glx2 Phe1 Gly4 HisO LleO Lys3 Leu2 MetO Pro4 Arg8 Ser2 Thrl
Val2 Tyr2, found A1a5.2 Asx4.8 CmCys0.6 G1x2.0 Phel.O G1y4.1 HisO LleO Lys2.9
Leu2.0 MetO Pro3.7 Arg6.9 Serl.8 Thr0.8 Va12.0 Tyrl.B; mass predicted 4786.4,
found
4787.1.
For deprotection and purification of PPEBP2sH, PAL resin (10 mg) containing
protected PPEBP2sH was allowed to react for seven hours at room temperature in
the
deprotection cocktail and the solvent was removed. Diethylether (1 ml) and
dithiothreitol (20 mg) were added, the supernatant was removed after
centrifugation
and the precipitate dried. The crude peptide was dissolved in 1 ml phosphate-
buffered
saline (pH 7.5) containing 5 mg fresh dithiothreitol and filtered. The peptide
was
purified by reversed phase HPLC (Vydac semipreparative~300 ~ C18, 5 microns,
10.0
x 250 mm) using a linear 120 minute gradient of 100 - 50% Buffer A in Buffer
B. The
peptide eluted at 67.8 minutes using a flow rate of 4 ml/min and was
characterized by
electrospray ionization mass spectrometry: mass predicted 4654.2, found
4653.6.
For preparation of PPEBP2sR, 0.070 mg of PPEBP2sH was dissolved in 0.50 ml
of 2 mg/ml (15 mM) 2-bromoacetamide in 20 mM sodium phosphate buffer (pH 7.5).
The reaction was allowed to proceed forty minutes at room temperature. The
peptide
was purified by reverse phase HPLC using a four minute linear gradient of 100 -
30%
Buffer A in Buffer B (Rainin analytical 100 ~ C18, 5 microns, 4.6 ~t 250 mm).
PPEBP2sH eluted at 24.9 minutes using a flow rate of 1 ml/min, and was
characterized
by electrospray ionization mass spectrometry and amino acid analysis. AAA
expected:
Alas Asx6 CmCys1 Glx3 Phel Gly4 HisO LleO Lys3 Leu2 MetO Pro4 Arg7 Ser2 Thrl
Val2 Tyrl, found A1a5.0 Asx5.8 CmCys0.9 G1x3.0 Phel.O G1y4.0 HisO L1e3.0
Lys3.0
Leu2.1 MetO Pro4 Arg7 Ser2 Thrl Val2 Tyrl; mass predicted 4711.3, fotmd
4710.8.
For deprotection and purification of EBP 1 sH, PAL resin (12 mg) containing
protected EBPIsH was allowed to react for six hours at room temperature in the
deprotection cocktail and treated as described for PPEBPIsR. The crude peptide
was
dissolved in 1 riml phosphate-buffered saline (pH 7.5) with added
dithiothreitol (5 mg)
and filtered. The peptide was purified by reversed phase HPLC (Vydac
semipreparative 300 A C18, 5 microns, 10.0 x 250 mm) using a 72 minute linear
gradient of 100 - 70% Buffer A in Buffer B. EBP 1 sH eluted at 49.6 minutes
using a
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flow rate of 1 ml/min and was characterized by electrospray ionization mass
spectrometry: mass predicted 3346.9, found 3346.2.
For preparation of EBPIsR, 150 micrograms of EBPIsH was dissolved in 0.50
ml of 2 mg/ml (15 mM) 2-cromoacetamide in 20 mM sodium phosphate buffer (pH
7.5). The reaction was allowed to proceed thirty minutes at room temperature.
The
peptide was purified by reverse phase HPLC (Rainin analytical 100 ~ C18, 5
microns,
4.6 x 250 mm) using a 40 minute linear gradient of 100 - 30% Buffer A in
Buffer B.
EBP 1 sR eluted at 17.0 minutes using a flow rate of 1 ml/min and was
characterized by
electrospray ionization mass spectrometry and amino acid analysis. AAA
expected:
Ala4 Asx3 CmCys1 Glxl Phel Gly2 HisO LleO Lys3 Leu2 MetO ProO ArgB Ser1 ThrO
Vall Tyrl, found A1a3.9 Asx3.0 CmCys0.9 G1x1.0 Phel.O G1y2.1 HisO LleO Lys2.8
Leu2.0 MetO ProO Arg6.9 Ser0.9 ThrO Va11.0 Tyrl.O; mass predicted 3404.0;
found
3403.7.
For C/EBPls2, a stock solution of the purified C/EBP peptide was prepared by
dissolution in phosphate-buffered saline with 10 mM dithiothreitol. The
solution was
heated to 95°C and allowed to slowly cool to room temperature in order
to assure
reduction of the cysteine near the carboxy terminus of the peptide. The
peptide was
then used immediately for EMSA analysis. The peptide was characterized by
amino
acid analysis. AAA expected: Ala8 AsxlB G1x18 PheS Gly6 HisO Lle4 Lysl4 Leul2
Met3 Pro6 Argl3 SerlS Thr7 Val9 Tyr2, found A1a9.2 Asx16.9 G1x18.0 Phe4.5
G1y7.0
HisO L1e3.8 Lys14.2 Leu11.3 Met2.7 Pro6.0 Arg10.8 Ser13.0 Thr7.0 Va18.0
Tyrl.7.
Example 2 - Binding of miniature proteins to DNA
Miniature protein-binding to DNA was measured using an electrophoretic
mobility shift assay performed in a Model SE600 Dual-Controller Vertical Slab
Unit
(Hoefer) using 14 x 16 cm gel plates. Temperature was controlled using a
constant
temperature bath. Reactions were performed in a binding buffer composed of 137
mM
NaCI, 2.7 mM KCI, 4.3 mM NaZHP04, 1.4 mM NaH2P04 (pH 7.4), 1 mM EDTA,
0.1% NP-40, 0.4 mg/ml BSA (non-acetylated) and 5% glycerol. For experiments
involving the bZIP peptide C/EBPis2, the binding buffer was supplemented with
2 mM
dithiothreitol. Serial peptide dilutions were performed as 1:1 dilutions with
binding
buffer. In general, 0.002 ml of gamma 32P-labeled, double-stranded DNA (CRE24,
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hsCRE24, C/EBP24 or hsCEBP24; final concentration <_ 50 pM in binding buffer;
final
concentration <_ 5 pM for peptides with Kapp < 500 pM) in binding buffer were
added to
0.008 ml of a serial peptide dilution on ice. Peptide-DNA mixtures were
incubated for
thirty minutes on ice and then applied to a pre-equilibrated, native
polyacrylamide gel
(8% acrylamide:bisacrylamide) prepared in 10 mM Tris buffer (pH. 8.1). Gels
were
allowed to run 0.75 to 1.5 hours at 500 V and were dried on a Model SE1160
Drygel
Sr. gel dryer (Hoefer). The gels were analyzed using a Storm 840
Phosphorimager
(Molecular Dynamics). Amounts of free and bound DNA were quantified and
analyzed using the program KaleidaGraph 3.0 (Synergy Software). Dissociation
constants were determined by fitting the data to the Langmuir equation = c[(1+
(Kapp/peptideTn))-y where n = 1 for PPEBPsR and EBPsR and n = 2 for C/EBPISZ.
In
these equations, theta = cpm in protein-DNA complex/(cpm in protein-DNA
complex +
cpm free DNA); peptideT = the total peptide concentration and c is an
adjustable
parameter representing the maximum value of theta (c <_ 1; for many peptides c
was
defined as 1). Values reported represent the average of at least three
independent trials
~ the standard error. Error bars on the plots represent the standard error for
each data
point.
For determination of binding stoichiometry, binding reactions were performed
in the same buffer used for EMSA experiments. Each reaction contained 200 nM
hsCRE24 and between 25 nM to 1600 nM PPEBP 1 sR. The hsCEBP24 concentration
was
determined by measuring the absorbance of each single stranded oligonucleotide
at 260
nm. One strand of each duplex was labeled with gamma-32P. A small amount
(0.010
ml) of labeled DNA was added to a 0.002 mM stock of the same strand. The
ensure
that the labeled strand annealed completely to its complement, an excess of
cold
complementary strand was added and the mixture was allowed to anneal by
heating to
95 °C for two minutes and slowly cooling to room temperature. Labeled
hsCEBP2a
was added to the PPEBP 1 sR solution and the reaction incubated at 4°C
for thii ty
minutes before being applied to a native 8% (80:1 acrylamide:bisacrylamide)
prepaxed
in 10 mM Tris buffer (pH = 8.0 at 4 °C). The gels were suspended in a
chamber
containing 10 mM Tris buffer that was kept at 4 °C by immersion in a
water-circulating
temperature bath. The gels were dried and quantified with a Phosphorimager
(Molecular Dynamics).
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No significant DNA binding was detected with peptides PPBROsR (SEQ ID NO:
8), PPBRIOsR (SEQ ID NO: 9) and PPBRIIsR (SEQ ID NO: 10) which lacked one or
more of these DNA-contact residues. High-affinity DNA binding was observed
with a
peptide that contained these three residues: The equilibrium dissociation
constant (I~)
of the PPBR2sR (SEQ ID NO: 11) binding to hsCRE was 5 nM under conditions of
physiological ionic strength. DNA affinity was enhanced further by selective
alanine
substitutions that increased the overall alpha-helical propensity of the
peptide,
producing the PPBR4sR-hsCRE24 complex whose I~ was 1.5 nM under identical
conditions. Formation of the PPBR4sR-hsCRE24 complex was unaffected by high
concentrations of poly (dIdC)-(dIdC) (Garner & Revzin, (1981) Nucl. Acids Res.
9,
3047-3048; Fried ~ Crothers, (1981) Nucl. Acids Res. 9, 6505-6506) or a
scrambled
CRE site (NON) indicating that the high stability of PPBR4sR-hsCRE24 was not
due
primarily to nonspecific ionic interactions. Circular dichroism experiments
indicated
that like bZIP peptides (Weiss et al., (1990) Nature 347, 575-578; O'Neil,
(1990)
Science 249, 774-778), no detectable changes in secondary structure occurred.
PPBR4sR (SEQ ID NO: 12) attained a fully alpha-helical conformation only in
the
presence of specific DNA (The CD spectrum of PPBR4sR was unchanged between
0.001 and 0.020 mM, indicating that no detectable changes in secondary
structure
occurred in this range. Addition of hsCRE DNA significantly increased the
alpha-helix
content of PPBR4sR while smaller changes were observed upon addition of hsCEBP
D,NA.
Although others have described monopartite DNA recognition by basic segment
peptides, the affinities reported have been only moderate (60 nM-0.003mM), and
the
complexes are stable only in very low ionic strength buffers (Park et al.,
(1996) J. Am.
Chem. Soc. 118, 4235-4239; Morii et al., (1996) J. Am. Chem. Soc. 118, 10011-
10012). PPBR4sR represents the first example of high affinity, monopartite,
major
groove recognition at physiological ionic strength.
Example 3 - Role of hydrophobic core in miniature protein-binding to DNA
The contribution of hydrophobic core formation on PPBR4sR-hsCRE24 complex
stability was examined utilizing LTV circular dichroism experiments. Circular
dichroism spectra were recorded in PBS on an Aviv-202 CD spectrometer and were
background corrected but not smoothed. Wavelength scans were performed at 4
°C
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between 200 and 260 nm at 1 nm intervals with a recording time of five seconds
at each
interval. Thermal denaturation curves were measured at 222 nm between 4
°C and 98
°C with 2 °C steps and one minute equilibration at each
temperature. Mean residue
ellipticity and percent helicity were calculated from the value at 222 nm
after
background correction.
G27 lacked the polyproline helix and turn, whereas PPBR4-deltasR contained D-
tryptophan at position four and leucine at position thirty-one. Modeling
studies
suggested that these substitutions would disrupt core formation by kinking the
polyproline or the alpha-helix. The stability of the G27-hsCRE24 and PPBR4-
deltasR-
hsCRE24 complexes were 3.1 and 3.2 kcal-mol-1 lower, respectively, than that
of
PPBR4sR-hsCRE24 complex. These data indicate that hydrophobic core formation
stabilized the PPBR4sR-hsCRE24 complex by as much as 3 kcal~mol'1.
Example 4 - DNA sequence specificity of miniature protein binding
The sequence specificity of PPBR4sR was examined by comparing its affinity
for hsCRE24 (SEQ ID NO: 13) to that for hsCEBP24 (SEQ ID NO: 4), a sequence
containing the half site recognized by C/EBP bZIP proteins (Agre et al.,
(1989)
Science 246, 922-926) using the electrophoretic mobility shift assay described
above.
This half site (ATTGC) differs from the CRE half site (ATGAC) by two base
pairs and
provides an excellent measure of base pair specificity (Suckow et al., (1993)
EMBO J.
12, 1193-1200; Johnson, (1993) Mol. Cell. Biol. 13, 6919-6930). PPBR4sR
displayed
remarkable specificity for hSCRE24. The specificity ratio Kre1
(I~(hsCRE)/I~(hsCEPB)) describing preferred recognition of hsCRE24 by PPBR4sR
was 2600 (delta,delta-G = -4.4 kcahmol'1). By contrast, G56 which comprised
the bZIP
element of GCN4, displayed low specificity. Specificity ratios of 118 and 180
were
observed for binding of CRE24 (SEQ ID NO: 3) by G56 in preference to CEBPz4
(SEQ
ID NO: 4) and hsCRE24 in preference to hsCEBP24 (delta,delta-G = -2.6 and -2.9
kcahmol'1, respectively). The relative specificities of G56 and PPBR4sR were
most
recognizable when one considered the concentration of each protein required to
bind
one-half of the two DNA. For PPBR4sR, this difference corresponded to a ratio
of
2600, whereas for G56, it corresponded to a ratio of eleven. PPBR4sR more
readily
distinguished the two base pair difference between hsCRE24 and hsCEBP2a than
Gss
distinguished CRE24 from hsCEBP24, two sequences that differed by six of ten
base
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pairs. These comparisons emphasize that PPBR4sR was considerably more
selective
than was GCN4, the protein on which its design was based.
Example 5 - Construction of synthetic genes encoding a miniature protein
As described into detail below, the phage display vector pJC20 was derived
from the monovalent phage display vector pCANTABSE (Pharmacia). pJC20 was
prepared by inserting a synthetic gene encoding aPP between the unique Sfi I
and Not I
restriction sites found in pCANTABSE. The synthetic aPP gene contained codons
for
optimal protein expression in E. coli and four restriction sites (Xma I, Age
I, Bgl II and
Pst I) absent in pCANTABSE. These restriction sites allow for the efficient
construction of genes encoding a variety of discrete miniature proteins as
well as for
the introduction of genetic diversity. The vector pJC21 was prepared by
inserting a
synthetic gene encoding residues 18-42 of PPBR4 between the unique Bgl II and
Not I
sites in pJC20. The identities of pJC20 and pJC21 were confirmed by automated
DNA
sequencing
A synthetic gene for aPP was constructed using codons chosen to optimize
expression in E. coli and incorporated four unique restriction sites to
facilitate cassette
mutagenesis. The 142 base pair duplex insert was generated by use of mutually
primed
synthesis and the oligonucleotides APP.TS (CTA TGC GGC CCA GCC GGC CGG
TCC GTC CCA GCC GAC CTA CCC GGG TGA CGA CGC ACC GGT TGA AGA
TCT GAT CCG TTT CTA CAA CGA CCT GCA GCA GTA CCT GAA CGT TGT
TAC CCG TCA CCG TTA CGC GGC CGC AGG TGC G) (SEQ ID NO: 39) and
APP.BS (CTA TGC GGC CCA GCC GGC CGG TCC GTC CCA GCC GAC CTA
CCC CGG GTG ACG ACG CAC CGG TTG AAG ATC TGA TCC GTT TCT ACA
ACG) (SEQ ID NO: 40) which overlap at nineteen base pairs. The reaction
mixture
(20 ml) contained 8 pmol APP.TS, 8 pmol APP.BS, 1x ThermoPol buffer (New
England Biolabs), 2 mg BSA, 1 mM dNTPs, 25 mCi [gamma-32P] ATP, 5 mM MgS04
and 2 ml Vent(exo-) DNA polymerase and was incubated at 94°C for thirty
seconds,
60°C for thirty seconds and 72°C for one minute. The major
reaction product was
purified from a denaturing (8 M urea) 10% acrylamide (29:1 acrylamide:bis-
acrylamide) gel and amplified by PCR in a 0.100 ml volume containing 1,500
pmol of
the primers CTA TGC GGC CCA GCC GGC CGG (SEQ ID NO: 41) and CGC ACC
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TGC GGC CGC GTA ACG (SEQ ID NO: 42), 0.010 ml template, 0.25 mM dNTPs, 5
mM MgS04, lat ThermoPol buffer (New England Biolabs) and 2 ml Vent(exo-) (New
England Biolabs). The PCR reaction was subjected to thirty cycles of
denaturation
(94°C for thirty seconds), annealing (60°C for thirty seconds)
and extension (72°C for
one minute). The insert was digested with Sfi I at 50°C in NEB buffer
two for four
hours. This buffer was then supplemented with NaCl to a final concentration of
100
mM and with Tris-HCl to a final concentration of 50 mM before digestion with
Not I
for four hours at 37°C. The resulting insert was ligated into the
vector pCANTAB-SE
(Pharmacia) in a reaction containing 800 units T4 DNA ligase (New England
Biolabs),
50 mM Tris-HCl (pH 7.~), 10 mM MgCl2, 10 mM DTT, 25 mg/ml BSA, 1 mM ATP,
250 ng pCANTABSE at 16°C for one and a half hours. The ligation
products were
transformed by electroporation into TGl E. coli and the resulting plasmid
designated
pJC20. A synthetic gene for PPBR4 was generated by replacing fifty-seven base
pair at
the 3' end of the aPP synthetic gene (in pJC20) with the sequence encoding the
C-
terminal twenty-five amino acids of PPBR4.
The oligonucleotides PPBR4TS (GAT CTG AAG CGC TTT CGT AAC ACC
CTG GCT GCG CGC CGT TCC CGT GCA CGT AAA GCT GCA CGT GCT GCA
GCT GGT GGT TGC GC) (SEQ ID NO: 43) and PPBR4BS (CGC ACC TGC GGC
CGC GCA ACC ACC AGC TGC AGC ACG TGC AGC TTT ACG TGC ACG GGA
ACG GCG CGC AGC CAG GGT GTT ACG AAA GCG CTT CAG ATC TTC AAC
C) (SEQ ID NO: 44) were annealed and phosphorylated on the 5' end to form the
PPBR4 insert. The PPBR4 insert was ligated into pJC20 that had been previously
digested with Bgl II and Not I and dephosphorylated with enzyme. The ligation
reaction mixture contained X00 units T4 DNA ligase in 50 mM Tris-HCl (pH 7.8),
10
mM MgCl2, 10 mM DTT, 25 mg/ml BSA, 1 mM ATP, 90 ng digested pCANTAB-SE
and 8 ng annealed insert. After reaction, the ligation mixture was transformed
into
electro-competent TG1 E. coli. The plasmid was designated pJC2l. The sequences
of
all final constructs were confirmed by automated sequencing.
Example 6 - DNA-binding miniature protein pha~;e library construction
A 10 ml volume of 2xYT containing 100 mg/ml ampicillin and 2% glucose was
innoculated with a 500 ml overnight culture of TG-1 E. coli containing the
plasmids
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pJC20 or pJC21 and shaken at 37 °C to an OD6oo = 0.8. 4 x 101°
pfu of M13 K07
helper phage were added and shaking continued for an additional one hour.
Cells were
pelleted for fifteen minutes at 5000 x g and resuspended in an equal volume of
2xYT
containing 100 mg/ml ampicillin and 50 mg/ml kanamycin and grown for ten hours
with shaking. Cells were pelleted by centrifugation at 5000 x g for twenty
minutes and
the phage supernatant filtered through a 0.45 micron filter before
precipitation with
PEG/NaCl (20% w/v PEG-8000, 2.5 M NaCI in ddH20) on ice for forty-five
minutes.
Phage were pelleted at 13000 x g for thirty minutes at 4°C and
resuspended in binding
buffer.
Example 7 - Expression of miniature proteins b~pha~e
As a first step towards displaying miniature proteins on the surface of phage,
the inventors sought to verify that aPP was' expressed from the synthetic
gene, which is
under the control of a lac promoter. To this end, TG-1 E. coli harboring pJC20
were
induced with isopropylthiogalactoside (IPTG), lysed and the cell lysates
probed with a
rabbit anti-aPP antibody (Peninsula Laboratories #RGG-7194) as described
below.
TG1 cells containing pJC20 were grown for one hour at 30°C in 2xYT
containing ampicillin at 100 mg/ml and 2% glucose. Cells were pelleted by
centrifugation at 5000 x g and resuspended in an equal volume of 2xYT
containing 100
mg/ml ampicillin and 1 mM IPTG, grown for three hours at 30 °C and then
lysed by
boiling in SDS sample buffer. Aliquots were loaded onto a Pharmacia Phast HOMO
20
gel and electrphoresed at 95 V until the solvent front ran off the gel.
Proteins in the gel
were transferred to an Immobilon-P membrane at 65 °C for one hour. The
membrane
was blocked for thirty minutes with TBST (20 mM Tris-HCl (pH 8.0), 150 mM
NaCI,
0.05% Tween-20) containing 0.5% BSA and then incubated with a 1:10000 dilution
of
rabbit anti-aPP (Peninsula Laboratories RGG-7194) provided at 4 mg/ml. The
membrane was then washed three times (five minutes per wash) with TBST and
then
incubated with TBST containing a goat anti-rabbit alkaline phosphatase
conjugate
(Santa Cruz sc-2007) at a 1:1000 dilution. After three five minute washes with
TBST
and a single wash with TBS (TBST lacking Tween-20), the membrane was stained
with
VISTRA ECF (Pharmacia) and visualized at 405 nm on a STORM 850 Phosphoimager
(Molecular Dynamics).
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For Western blots on phage particles, 10 ml of phage were produced and
precipitated with PEG/NaCI as described above. The phage were then resuspended
in 1
ml ddH20, precipitated with 200 ml of PEG/NaCl, resuspended in 100 ml ddH20
and
heated to 95 °C in SDS sample buffer for ten minutes. The phage
proteins were then
applied to a 10% SDS gel (29:1 acrylamide:bisacrylamide) and subjected to
electrophoresis at 20 mA in Tris-glycine electrophoresis buffer until the
solvent front
ran off the gel. The separated proteins were transferred to an Immobilon-P
membrane
(Millipore) at 20 V for four hours using a TE62 unit (Pharmacia) containing
Towbin
buffer (20% MeOH, 25 mM Tris-HCl (pH 8), 192 mM glycine, 0.1% SDS (w/v)) at 4
°C. After blocking with 5% nonfat mills in TBST for sixteen hours and
washing twice
(five minutes per wash) with TBST, the membrane was probed for thirty minutes
with
anti-aPP in TBST supplemented with 2.5% nonfat milk. The membrane was washed
three times (five minutes per wash) with TBST, then exposed to a goat anti-
rabbit
antibody-alkaline phosphatase conjugate (Santa Cruz sc-2007) at a 1:5000
dilution in
TBST supplemented with 2.5% nonfat milk for fifteen minutes. After washing
three
times (five minutes per wash) with TBST and two times (five minutes per wash)
with
TBS the membrane was stained with VISTRA ECF (Pharmacia) and visualized at 405
nm on a STORM 850 phosphorimager (Molecular Dynamics).
These experiments demonstrate clear evidence for IPTG-inducible expression
of aPP fused to the minor capsid protein III of M13 bacteriophage. To
investigate
whether this fusion protein was assembled into viable phage particles,
purified phage
were, phage proteins resolved using SDS-PAGE and probed with the rabbit anti-
aPP
antibody. The Western blot clearly shows that the fusion protein containing
aPP and
protein III is incorporated into fully assembled M13 phage particles. No
signal was
observed when phage produced from pJC21 bearing cells were probed with the
rabbit
anti-aPP antibody
Example 8 - Functional selection of DNA-binding miniature proteins on phase
As a first step towards the optimization of PPBR4, the inventors confirmed
that
phage displaying PPBR4 could be selected over phage bearing aPP when sorted on
the
basis of specific DNA-binding. Phage displaying either PPBR4 or its progenitor
aPP
were panned against magnetic beads coated with a twenty-four base pair duplex
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oligonucleotide containing the five base pair sequence recognized by PPBR4,
half site
CRE (hsCRE, ATGAC). The DNA was attached to streptavidin coated beads through
a
3' biotin TEG (triethyleneglycol) linker (Glen Research). Panning was
performed
essentially as previously described and as set forth below (Choo 8z Klug,
(1994) Proc.
Natl. Acad. Sci. USA 91, 11163-11167).
For panning experiments, 0.5 mg of streptavidin-coated M-280 magnetic beads
(Dynal) were washed six times with 50 ml of 2x B+W buffer (10 mM Tris-HCl (pH
7.5), 1 mM EDTA, 2.0 M NaCl). Each wash step was performed for two minutes.
The
beads were blocked by incubation in 50 ml of lie B+W containing 6% nonfat milk
for
fourteen hours. The beads were then washed five times with 50 ml of 1x B+W and
resuspended in 50 ml of lx B+W containing approximately 1 mM duplex hsCRE242
carrying a 3' biotin label on one strand for twelve minutes. This procedure
loaded
approximately 75 pmol DNA per mg bead. The beads were then washed five times
with 50 ml of phage binding buffer (phosphate buffered saline supplemented
with 0.4
mg/ml BSA, 0.1 % NP-40 and 2.5 mg of poly-dIdC). 1010 phage in a volume of 0.4
ml were added to the beads at 4 °C and incubated with rotation on a
Labquake shaker
rotisserie for two hours. Beads were washed five times for five minutes at 4
°C with
wash buffer (phage binding buffer lacking poly-dIdC). Bound phage were eluted
by
the addition of wash buffer containing 4 M NaCl and an increase in temperature
to 25
°C for two hours. 200 ml of the elution and 200 ml of phage not subject
to panning
were used to infect 7 ml of log phase TG-1 E. coli. After one hour, serial
dilutions of
infected cells were plated on SOBAG (SOB media supplemented with ampicillin to
100 m~/ml and 2% glucose) and grown for twelve hours at 30 °C. Values
of percent
retention were calculated where percent retention = (output titer/input titer)
~e 100.
In the present experiments, wash conditions were optimized to maximize
differential retention of phage displaying PPBR4 and phage displaying aPP. In
phosphate buffered saline (PBS) supplemented with 0.1% NP-40, 0.4 mg/ml BSA
and
2.5 ~,g/ml poly-dIdC, the percent retention of PPBR4 phage on hsCRE beads was
ten
times greater than that of aPP phage. This result indicates that miniature
proteins
generated by protein grafting can be functionally selected on M13 phage.
Example 9 - Isolation of highly selective DNA-binding miniature proteins
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Two phage libraries were created essentially as described in the previous
examples to identify appropriately folded PPBR4 analogs that would bind with
higher
affinity and specificity. The members of libraries A and B differ from PPBR4
at three
(library A) or four (library B) positions on the PPII helix. The proline
residues retained
at positions two and five of library A are highly conserved among PP-fold
proteins. It
was anticipated that retention of these two prolines would effectively
constrain the
conformational space available to library A members and that most would
contain N-
terminal PPII helices. Such conformational constraints are absent in library
B,
acknowledging that there may be many ways to stabilize DNA-bound alpha-
helices.
Since the amino acids at positions two and five of library B are not
restricted to
proline, it was anticipated that this library would sample a larger fraction
of available
phi-psi space. Phage were sorted for three rounds on the basis of their
ability to bind an
oligonucleotide duplex containing the sequence ATGAC (hsCRE). To favor
identification of sequences that bound hsCRE with high affinity at ambient
temperature, two rounds of selection at 4°C were followed by a single
round at room
temperature. By the final round, library A phage were retained at a level only
comparable to PPBR4 phage and were not considered further. Library B phage
were
retained at a level comparable to PPBR4 phage after the first round, but at
levels fifteen
to sixteen times better than PPBR4 phage after the subsequent two rounds.
Twelve
library B clones were sequenced after round three. Six sequences (p007, p009,
p011,
p012, p013, and p016) were synthesized and the DNA-binding properties of four
analyzed in detail.
Quantitative electrophoretic mobility shift experiments were performed as
described in the previous examples to assess the DNA affinities of p007, p011,
p012,
and p016. All peptides tested bound hsCRE as well or better than did PPBR4 or
G27
(the isolated basic region of GCN4). At 4°C, p011 and p012 bound hsCRE
with
affinities of 1.5 ~ 0.2 nM and 2.5 ~ 0.5 nM, whereas p016 bound hsCRE with an
affinity of 300 ~ 60 pM. Of particular interest is p007, which bound hsCRE to
form an
exceptionally stable complex with a dissociation constant of 23 ~ 1.2 pM. This
peptide
bound specific DNA approximately 100-times better than did PPBR4 (I~ =1.9 ~
0.2
nM) and approximately 20,000 times better than did G27 (I~d = 410 ~ 53 nM).
Moreover, at 25°C p007 bound hsCRE with an affinity of 1.6 ~ 0.1 nM.
Neither
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PPBR4 nor G27 showed evidence of DNA binding at this temperature. P007 binds
specific DNA considerably more tightly than two fingers from the Tramtrack
zinc
finger protein, which binds five base pairs of DNA with an affinity of 400 nM
(Segal &
Barbas, (2000) Curr. Op. Chem. Biol. 4, 34-35).
Example 10 - Specificity of hi~hl~selective miniature protein DNA-binding
The specificity of DNA binding was investigated by determining the affinity of
p007 for several duplex oligonucleotides containing two base pair changes
within the
five base pair hsCRE sequence using quantitative electrophoretic mobility
shift assays
as described in the previous examples. p007 was extremely discriminating,
exhibiting
a specificity ratio R (defined as the ratio of the dissociation constants of
specific and
mutated complexes) between 200 and 800 (delta,delta-G = -3.3 to 4.0 kcal mol-
1). This
high level of discrimination was observed across the entire five base pair
hsCRE
sequence, indicating that no single interaction dominated the free energy of
the p007-
hsCRE complex and that the binding energy is partitioned across the entire
protein-
DNA interface. By contrast, at 4°C PPBR4 discriminates poorly
(delta,delta-G = -1.7
kcal
mol-1) against sequences possessing mutations at the 5' terminus of hsCRE.
To investigate the possibility that DNA sequences other than these four might
bind p007 tightly, the affinity of p007 for calf thymus DNA (CT DNA) which
possesses a potential binding site in every register on either DNA strand was
measured.
The average specificity ratio for recognition of hsCRE in preference to any
site in CT
DNA was 4169. This ratio is considerably greater than the number of potential
competitor sites (45 = 1024). Whereas the triple zinc finger construct Zif268
and
variants thereof selected by phage display fail to uniquely specify one to two
base pairs
of their nine base pair binding sites (Li et al., (1992) Biochemistry 31, 1245-
1253),
p007 completely specifies all five base pairs of its target sequence. In fact,
even if each
possible five base pair competitor site were present at equal molarity to the
taxget site,
80% of the p007 molecules would be bound to hsCRE, despite the effects of mass
action.
Example 11- NMR characterization of miniature protein structure
For NMR Spectroscopy, p007 was dissolved in 90% H20/10% D20 containing 4
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mM KCl, 205 mM NaCI, 6.5 mM Na2HP04, 2.1 mM KHZP04 (pH 7.4). Peptide
concentration was approximately 1.5 mM. Chemical shifts were referenced in ppm
from internal 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt. All
spectra were
recorded on a Varian 800 MHz Inova instrument at 2 °C with a sweep
width of 9000
Hz. NOESY experiments were performed using a waterflip-watergate pulse
sequence
for water suppression with 4096t2 x SOOt1 complex points. Mixing times of 50,
150
and 300 ms were acquired. DQF-COSY spectra (60 ms mixing time) were acquired
with 2048t2 x 300t1 complex points. Data was processing was performed on a
Silicon
Graphics Workstation using Felix 98 (MSI). Prior to Fourier transform of the
free
induction decays, a gaussian window function was applied to NOESY spectra,
while a
Kaiser window function was applied to DQF-COSY spectra. The digital resolution
of
the NOESY spectra was 2.2 Hz/pt. DQF COSY data was zero filled to yield a 8192
x
8192 matrix with a digital resolution of 1.1 Hz. Spectra were assigned by
standard
methods.
Multidimensional NMR experiments allowed for characterization of the
structure of p007 in greater detail. The backbone and side-chain
connectivities in p007
were assigned on the basis of reasonably disperse NOESY spectra. The presence
of
amide-amide cross peaks between residues at positions i and i+3 and i and i+4
defined
an alpha-helical conformation for residues 14-30. Eleven long range NOEs
between
residues 8 and 17, 8 and 20, 7 and 20, 5 and 20, 4 and 27, 2 and 29, 2 and 30
specify a
folded structure that superimposes on residues 5-8 and 15-28 of aPP with a
backbone
rmsd of 1.61. Thus, the main chain folds of p007 and aPP are remarkably
similar,
with residues 5, 7 and 8 proximal to residue 20 and residues 1 and 2 proximal
to
residue 30. As in previous studies of pancreatic fold polypeptides (Blundell
et al.,
(1981) 78, 4175-4176), the PPII helix proposed for residues 1-8 of p007 is
under-
defined by the NMR data. However, in light of the similarity between the aPP
and
p007 folds, p007 must contain a structure similar to a PPII helix.
Example 12 - Protein-binding miniature protein phage library construction
For construction of the aPPBAK library, mutagenesis was carried out using the
NNS codon scheme, where N = any base and S = G/C. This scheme codes for all
twenty amino acids and the amber stop codon TAG which is suppressed by
insertion of
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glutamine in the E. coli SupE strains used. The oligonucleotides BAKLIB: GGT
GAC
GACGCA CCG GTT GAA GAT CTG ATC CGC TTT GTT NNS CGT CTG CTG
NNS TAC ATC NNS GAC NNS ATC AAC CGT CGT GCG GCC GCA GGT GCG
(SEQ ID NO: 45) and PBAI~LIB: CGC ACC TGC GGC GGCACG ACG (SEQ ID
NO: 46) were synthesized and purified by denaturing gel electrophoresis. 400
pmol of
each oligonucleotide were annealed in lx Sequenase buffer (USB) in a total
volume of
0.20 ml. The annealed oligonucleotides were converted to duplex DNA by primer
extension upon addition of 2.5 mM dNTPs, 1 mglml BSA and 50 units Sequenase
(USB) and incubation at 37°C for thirty minutes. The duplex DNA was
digested in 1x
buffer 3 (New England Biolabs) by the addition of 0.015 ml Bgl II, 0.015 ml
Not I, 2.5
mM DTT, 0.1 mg/ml BSA in a total volume of 0.430 ml. The reaction mixture was
extracted twice with an equal volume of Tris buffered phenol (pH 8.0) and
applied to a
15% acrylamide (29:1 acrylamide:bisacrylamide) gel in 1x TBE at 500 V. The
doubly
digested product was visualized by ethidium staining, excised and extracted in
1 x TE.
The insert was ethanol precipitated. 0.12 mg of the vector pJC20 was digested
with
0.05 ml of Bgl II, Not I and Pst I in a total volume of 0.60 ml. The digested
vector was
purified by Chromaspin 1000 size exclusion chromatography (Clonetech) and
phenol
chloroform extraction followed by ethanol precipitation. Ligations were
performed
using the Ligation express kit (Clontech) with 830 ng of vector (pJC20) and 14
ng of
insert. Transformation by electroporation in to TG-1 E. coli yielded 3 x 106
transformants. The number of transformants is greater than the theoretical
diversity of
the library (324 = 1.05 ~ 106) and the library is statistically greater than
90% complete.
Automated DNA sequencing of twenty clones showed the mutant genes were
inserted
correctly in all cases.
Example 13 - Functional selection of protein-binding miniature proteins on
phase
For biopanning of the aPPBAK library, a glutathione coated microtiter plate
(Reacti-bind glutathione coated plate #15140, Pierce) was washed three times
with 0.20
ml of PBS per wash. Human recombinant Bcl-2 (1-205) was obtained as a soluble
GST-fusion from Santa Cruz Biotechnology. 9.0 pmol of Bcl-2 in 0.20 ml of PBS
was
added to each well and incubated at 4°C for twelve hours with shaking.
The wells were
then blocked for three hours with 0.20 ml of TBST containing 5% nonfat dry
milk.
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Before use, the well was washed three times with TBST for five minutes per
wash.
Phage were produced, harvested and propagated as described in the previous
examples, with the exception that, in rounds three through five, XL1-blue
cells were
used instead of TG-1 cells to propagate phage particles. This change
eliminated
problems encountered previously with deletions in later rounds of selection,
which are
attributed to the Rec A+ nature of TG-1 E. coli. Phage particles were
resuspended in 2
ml of TBST. 0.20 ml of phage (1 x 101° particles) were added to each
well and
incubated for three hours at 4 °C in the first two rounds of selection
and at 25 °C in the
final three rounds. The wells were then washed ten times with 0.20 ml of TBST,
two
minute washes in the first round and five minute washes in subsequent rounds.
Washes
were performed at the same temperature in the binding reaction. After five
rounds of
selection, sixteen clones were sequenced by automated DNA sequencing.
The phage library BAKLIB was subjected to five rounds of panning against
immobilized GST-Bcl-2. The percent retention of the phage library increased
225-fold
over the course of the selection from 0.01 % in the first round to 2.25% in
the fifth
round. This increase in retention underestimates the improvement of library
retention
because the final round was carried out at 25 °C while the first round
was performed at
4 °C. After five rounds sixteen phagemid library clones were sequenced.
The selected
sequences (Fig. 1) show a high degree of convergence. Seven distinct sequences
were
isolated with four sequences represented multiple times. Interestingly,
residue 28 in the
library, which corresponds to I81 of Bak, is mutated to F in eleven of sixteen
round five
clones, although it was fixed in the initial pool. This result indicates that
within the
context of the scaffold, F28 is better at binding into the hydrophobic pocket
of Bcl-2
than I28. Eleven of sixteen sequences contain glycine at positions 75 and 82
as in Bak.
Indeed, one sequence that was represented two of sixteen times contained
residues
identical to those of Bak at all four randomized positions, this sequence
however, also
contained the I-F mutation at position 28. Comparison of the selected
sequences to
other BH3-containing proteins reveals further similarities. For example, at
position 26
of the library, R occurred in seven of the sixteen sequences and R or K is the
preferred
amino acid at this position (residue 79 in Bak) in most BH3 domains.
Similarly, an E
at position 31 of the library was selected in six of sixteen sequences, where
E/D is the
preferred amino acid at the corresponding position of most known BH3 domains.
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The similarities of selected amino acids at these positions to those in Bak
and
other BH3 domains indicates that the sequences of BH3 domains arose from the
requirement to bind Bcl-2 family proteins and not for other biological
function.
Further, it also indicates that the selected peptides bind Bcl-2 in the same
hydrophobic
pocket as does Bak. Interestingly, one sequence represented twice contained a
threonine at position 31 of the library. This residue provides both the methyl
group of a
valine which could contribute to hydrophobic core formation and a hydroxyl
group that
could provide a hydrogen bond acceptor like the native D/E residue in BH3
domains.
One sequence that appeared twice in the round five clones sequenced contained
a single
amino acid deletion with respect to the library design that places both the
aPP folding
residues and the Bcl-2 residues out of register.
Example 14 - Synthesis of~rotein-binding miniature proteins
Peptides were synthesized on a 0.10 mM scale using Fmoc chemistry. Each
peptide contained a free N-terminal amine and a C-terminal amide. Peptides
were
purified by reverse phase HPLC as described in the previous examples. Two sets
of
peptides were prepared, peptides 4099-4102 and the Bak peptide (SEQ ID NO:
73).
Peptides for fluorescent labeling and subsequence I~ determinations contained
an
additional carboxy-terminal YC sequence (the Y is derived from the native
sequence of
Bak), the cysteine of which was labeled with 5-iodoacetamidofluorescein
(SIAF).
Peptides at a final concentration of 200-400 mM were alkylated on the sulfur
atom of
C-terminal cysteines by incubation with ten equivalents of SIAF (Molecular
Probes) in
0.20 ml of a 50/50 mixture of DMF and PBS. The labeling reaction was performed
in
the dark for six hours at room temperature. Alkylation was essentially
quantitative as
judged by HPLC. Labeled peptides were purif ed by reverse phase C-18 HPLC. The
identifies of the peptides were verified by MALDI-TOF mass spectrometry
(Voyager,
Perseptive Biosystems). The molecular weights were as expected: p4099
theoretical
[MH+] = 3907, observed [MH+] = 3907; p4100 theoretical [MH+] = 4020, observed
[MH+] = 4020; p4101 theoretical [MH+] = 3921, observed [MH+] = 3922; p4102
theoretical [MH+] = 3901, observed [MH+] = 3902; Bak 72-94 theoretical [MH+] _
1724, observed [MH+] =1723; p4121-flu theoretical [MH+] = 4562, observed [MH+]
= 4560; p4122 theoretical [MH+] = 4675, observed [MH+] = 4766; p4123
theoretical
[MH+] = 4576, observed [MH+] = 4577; p4124 theoretical [MH+] = 4556, observed
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[MH+] = 4556; Bak-flu theoretical [MH+] = 2535, observed [MH+] = 2535. Peptide
concentrations were determined by amino acid analysis.
Example 15 - Binding of miiuature proteins to other proteins
To measure the equilibrium dissociation constant of Bcl-2 binding to the
selected peptides or the Bak BH3 peptide, Bcl-2 was serially diluted from
0.0036 mM
in PBS with the fluorescently labeled peptide added at a constant
concentration
between 0.020-0.040 mM. After equilibration for forty minutes at 4 °C,
the fluorescein
was excited at 492 ilm using a PS-220B lamp power supply (Photon Technologies)
and
the fluorescence emission spectra between 505 and 560 nm recorded on an 814
photomultiplier detection system (Photon Technologies) with a 2 nm stepsize
and a one
second equilibration time, using 5 nm slit widths. The fluorescence emission
maxima
at 515 nm for three independent trials were averaged and the dissociation
constants
calculated as previously described. Similar experiments were used to determine
the
dissociation constants for the Bak peptide or selected peptides binding
carbonic
anhydrase II (Sigma) or calmodulin (Sigma). The calmodulin binding was
measured in
a buffer composed of 20 nM HEPES (pH. 7.2), 130 mM KCI, 1 mM CaCl2 while
carbonic anhydrase binding was measured in PBS.
The Bak peptide along with four sequences represented multiple times in the
sixteen sequenced clones from round five were chemically synthesized. Bcl-2
binding
affinity of the peptides was determined by measuring the change in
fluorescence
emission of a carboxy-terminal fluorescein label on the peptide as a function
of Bcl-2
concentration. To validate this assay the Kd for the Bak peptide binding to
Bcl-2 was
measured. This Kd was 363 nM ~ 56 nM, consistent with a Kd of 340 nM
previously
reported for the Bak peptide Bcl-XL interaction (measured by fluorescence
quenching
of intrinsic tryptophan in Bcl-XL) and a Kd of about 200 nM reported for the
Bak Bcl-2
interaction (measured by fluorescence polarization of a fluorescein labeled
Bak
peptide). The Kd for the selected peptides were: p4099 Kd = 352 ~ 33 nM, p4100
Kd =
401 ~ 40 nM, p4101 I~ = 811 ~ 20 nM, p4102 3700 ~ 1400 nM. The Kd for all the
peptides without deletions indicate that they bind significantly better than
the mutant
p4102 that contains a deletion in the alpha-helix. Within this series of
peptides, p4099
(GAGT) binds about two-fold better than p4101 (GAGD), that differs in only a D
to T
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mutation at position 31. p4100 (GRGE) binds with comparable affinity to p4099
indicating that these two peptides represent convergent and equal solutions to
forming a
protein-protein interface.
In order to compare the specificity of 4099 to the Bak peptide, their
interaction
with Calmodulin was investigated. Calmodulin is known to bind a range of alpha
helices and Carbonic anhydrase II, which has a large hydrophobic cavity. p4099
bound
Calmodulin with a K.~ of 0.025 ~ 0.004 mM, while the Bak peptide bound
Calmodulin
with a K.~ of 0.025 ~ 0.004 mM. p4099 bound Carbonic anhydrase II with a I~ of
0.0086 ~ 0 mM, the Bak peptide bound Carbonic anhydrase with a I~ of 0.022 ~
0.0046 mM. p4099 discriminates well against these non-specific proteins
indicating
that the interaction between the peptide and Bcl-2 results from a
stereospecific set of
VanderWaals contacts.
Example 16 - Structure of protein-bindin~Lniniature proteins
Circular dichroism spectra were recorded in PBS on an Aviv 202 CD
Spectrometer and were background corrected but not smoothed. Wavelength scans
were performed at 4 °C between 200 and 260 nm at 1 nm intervals with a
recording
time of five seconds at each interval. Bak (72-94), 4099, 4100, 4101, 4102
were used
at concentrations of 0.028 mM, 0.0069 mM, 0.0119 mM, 0.014 mM and 0.016 mM
respectively. Thermal denaturation curves were measured at 222 nm between 4-98
°C
with 2 °C steps and one minute equilibration at each temperature.
Peptides were used
at the highest concentrations used for the wavelength scans described above.
Mean
residue elliptcity and percent helicity were calculated from the value at 222
nm after
background correction.
The structure of peptides was investigated by far UV circular dichroism as
described above. Wavelength scans reveal the previously reported random coil
signature for the Bak peptide. In contrast the selected peptides 4099, 4100,
4101, 4102
show minima at 208 and 222 nm, characteristic of alpha-helical content. The
mean
ellipticity of peptide 4099 was shown to be concentration independent down to
the
lowest concentration measurable 0.0011 mM. The percentage helicity of p4099 is
approximately 60%, consistent with an aPP-like tertiary fold in which residues
14-35
adopt a helical confirmation. This helicity is comparable to that seen for
p007, a
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peptide evolved to bind DNA with high affinity and specificity as described in
the
previous examples. Thermal denaturation of the peptides was monitored by far
UV
circular dichroism at 222 nm. p4099 had a cooperative thermal melt with a T,~
of
approximately 65 °C, comparable to the Tm reported for aPP.
Example 17 - Miniature proteins for inhibiting MDM2-p53 interactions
MDM2 is the principal cellular antagonist of the tumor suppressor protein p53
(Wu et al., J. Genes l~ev. 1993, 7, 1126). MDM2 antagonizes p53 function by
sequestering the p53 transcriptional activation domain and targeting it for
ubiquitin-
dependent degradation by the 26S proteasome. Elevated MDM2 levels are found in
a
variety of solid tumors containing wild type p53 and there is considerable
interest in
MDM2 ligands capable of up-regulating p53 activity in vitro or i~z vivo. The
high-
resolution structure of the MDM2~p53 activation domain peptide (p53AD) complex
reveals an irregular p53AD a-helix nestled into a deep, hydrophobic, MDM2
cleft
(Kussie, et al., Science 1996, 274, 948). This structure, along with
accompanying
mutagenesis data, suggests that complex stability (I~ = 600 nM) derives
predominantly
from interactions between three p53AD residues (F19, W23, and L26) and several
residues lining the MDM2 cleft.
Residue-by-residue alignment of the a-helical segments of p53 and aPP (Figure
2B) positions the three critical MDM2 contact residues (F19, W23, and L26) and
the
five important aPP folding residues (L14, F17, L21, Y24, L25) on the solvent-
exposed
and solvent-sequestered faces, respectively, of the aPP a-helix. Five
remaining a-
helical residues were varied across all twenty amino acids to (1) foster
additional
interactions with MDM2; (2) sustain the aPP fold; and (3) acknowledge the
imperfect
phi and psi angles found within p53AD bound to MDM2 (I~ussie, et al., Scie~zce
1996,
274, 948). The M13 phage library prepared contained 6 x 107 unique
transformants,
insuring that it would evaluate DNA sequence space with > 83% confidence.
Three
rounds of selection for binding GST-MDM21 immobilized on glutathione-coated
microtiter plates2 led to a 100-fold enrichment in affinity. Several peptides
from
rounds 2 and 3 (Figure 2B) were synthesized with a cysteine residue at the C-
terminus
and labeled with 5-iodoacetamidofluorescein to facilitate fluorescence
polarization
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analysis of MDM2 affinity (all synthetic peptides were purified to homogeneity
by
HPLC and characterized by MALDI-TOF mass spectrometry and amino acid
analysis).
Fluorescence polarization analysis indicated that all selected miniature
proteins
bound GST-MDM2 in the nanomolar concentration range (Figure 3). pP53-O5, in
particular, bound GST-MDM2 to form a complex with an equilibrium dissociation
constant (Kd) of 99 ~ 11 nM, a value that is significantly more favorable than
that of the
p53AD~MDM2 complex (Kd = 261 ~ 59 nM) (Kussie, et al., Science 1996, 274,
948).
Thus pP53-O5, which contains only 31 residues, binds MDM2 as well or better
than
109 residue thioredoxin derivatives that present p53AD (and variants thereof)
on an
active site loop (Bottger, et al., J. Med. Chem. 2000, 43, 3205). The CD
spectrum of
pP53-OS (2.75 ~,M) was characterized by negative ellipticity at 208 and 222 nm
that
was comparable to that of aPP and underwent a cooperative melting transition
(Tm) at
47 °C (Figure 4).
The specificity of pP53-OS was evaluated by measuring its affinity for several
receptors and enzymes that bind helical or hydrophobic peptides or small
molecules
(Figure 3). Calmodulin, an EF hand protein known for its ability to bind many
a-
helical peptides and proteins (Meador, et al., Science 1991, 257, 1251), bound
pP53-OS
in the high micromolar concentration range (Kd > 275 ~.M). Similar Kd values
characterized the affinity of pP53-OS for the bZIP region of Fos, which forms
dimeric
complexes with other bZIP proteins (42 ~,M) (Glover, et al., Nature 1995, 373,
257),
for carbonic anhydrase, which binds C02 (298 ~M) (Liljas, et al., Nat. New
Biol. 1972,
235,131), and for protein kinase A, which binds the a-helical peptide
inhibitor pKI (16
~,M) (Knighton, et al., Scietace 1991, 253, 414). The large difference (DOG =
2.8 - 4.4
kcal~mol-1) between the stabilities of these complexes and that of pP53-OS~GST-
MDM2 suggests that the latter is stabilized by highly stereospecific van der
Waals
interactions whose energetic benefit exceeds that available through non-
specific protein
contacts.
To establish whether pP53-OS bound MDM2 in a manner that would inhibit
binding of p53AD, we incubated GST-MDM2 and p53AD-Flu with varying
concentrations of pP53-OS and monitored the fraction of p53AD-Flu bound at
equilibrium (Figure 3). In the absence of pP53-O5, 60% of p53AD-Flu is bound
to
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GST-MDM2 under these conditions. Addition of pP53-OS led to a concentration-
dependent decrease (Ki = 722 nM) in the fraction of p53AD-Flu bound to GST-
MDM2.
Similar Ki values were determined at shorter and longer incubation times,
confirming
that equilibrium had been reached. By comparison, competition of p53AD-Flu by
unlabeled p53AD was characterized by K; =1.2 ~.M.
In conclusion, we have shown that protein grafting, in combination with
functional selection, provides rapid access to miniature protein ligands for
globular
protein surfaces. The molecules we describe possess affinities in the
nanomolar
concentration range and effectively discriminate against other proteins, even
those that
bind non-selectively to other helical, hydrophobic proteins. The combined
features of
high affinity, high selectivity and a compact protein fold should enhance the
utility of
miniature proteins for a wide variety of bioengineering and proteomics
applications
(Zhang, et al., Nat. Biotech. 2000,18, 71).
Example 18 - Miniature proteins for inhibitin~protein kinase A
The design of selective protein kinase inhibitors remains a significant
challenge
(Bridges, et al., J. Chem Rev 2001, 101, 2541-72; Scapin, et al., Drug Discov
Today
2002, 7, 601-11) because of both sheer numbers (more than 500 different
kinases are
found in a mammalian cell) and the highly conserved nature of the ATP binding
site
(Miller, W. T. Nat Struct Biol 2001, 8, 16-8). Only a small number of
selective kinase
inhibitors are known (Bridges, et al., J. Chem Rev 2001, 101, 2541-72; Cohen,
et al.,
Curr Opin Chem Biol 1999, 3, 459-65; Zimmermann, et al., Bioorg Med Chem Lett
1997, 7, 187-92).
The indolocarbazole natural product K252a is a potent, active-site directed
inhibitor of many tyrosine and serine/threonine kinases and a common starting
point
for the discovery of specific kinase inhibitors (Kase, et al., J Antibiot
1986, 39, 1059-
65; Kase, et al., Biochem Biophys Res Commun 1987, 142, 436-40; Hashimoto, et
al.,
Biochem Biophys Res Comm 1991, 181, 423-9; Tapley, et al., Oncogene 1992, 7,
371-
81). We have described a miniature protein design strategy in which the well-
folded
helix in avian pancreatic polypeptide (aPP) presents short a-helical
recognition
epitopes. The miniature proteins designed in this manner recognize even
shallow clefts
on protein surfaces with nanomolar affinities and high specificity (see, e.g.,
Examples
19-20). Here we demonstrate that designed variants of aPP can also impose
specificity
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on the potent but otherwise non-selective kinase inhibitor K252a by
recognizing non-
conserved features of the protein surface surrounding the ATP binding pocket.
Our
results suggest that bifunctional molecules that embody elements of protein
surface
recognition could represent a viable general strategy for selective kinase
inhibition.
Our design began with the structure of the catalytic subunit of cAMP-dependent
protein kinase (PKA) in complex with PKIS_z4, a peptide representing the
active portion
of the heat-stable Protein Kinase Inhibitor protein (Glass et al., J Biol Chem
1989, 264,
8802-10;
Zheng, et al., Acta Cryst 1993, D49, 362-5) (Figure 5). In this complex, the
PKIS_24 C-
terminal pseudosubstrate (residues 17-24) occupies the peptide substrate-
binding site
with energetically significant contacts from R18, R19, and I22 and R15 from
the
adjacent turn (residues 15-16); the N-terminal alpha helix (residues 5-13)
nestles in a
shallow hydrophobic groove outside the substrate-binding site with an
energetically
significant contact from F 10. Two separate alignments of the sequences of the
aPP and
PKIS_24 a.-helices were considered (Figure 6). Both alignments retain F10 of
the PKIS_z4
a-helix, all three pseudosubstrate contacts, and all residues required to
maintain the aPP
fold; alignment #1 also retains R15. The resulting molecules, 1 and 2, were
synthesized using standard solid phase methodology. A cysteine residue added
to the
C-terminus was modified with 5-iodo-acetamidofluorescein to facilitate
fluorescence
polarization analysis of PKA affinity.
The relative affinities of 1F1", 2Flu, and PKIS_24F1u for the catalytic
subunit of PKA
were measured by fluorescence polarization analysis in the presence and
absence of
ATP (Figure 7). In the presence of 100 ~M ATP, the complex between PKA and
lFlu
was characterized by an equilibrium dissociation constant (Kd) of 99 ~ 39 nM
(Figure
7a). The stability of PKA~1F1° was only 3-fold lower than that of
PKA~PKIS_z4Fi" under
identical conditions (Kd = 31 ~ 8 nM). 2rlu bound PKA with much lower affinity
(Kd =
570 ~ 123 nM), perhaps because it lacked R15, and was not considered further.
Surprisingly, lFiu retained significant affinity for PKA in the absence of ATP
(Kd = 230
~ 34 nM) (Figure 7b). By contrast, PKIS_24F1u bound PKA far more poorly in the
absence of ATP, as expected (Whitehouse, et al., J Biol Chem 1983, 258, 3693-
701),
showing a 50-fold decrease in affinity (Kd = 1.6 ~ 0.4 ~.M). Previous
structural and
biochemical studies have documented the dramatic change in PKA conformation
induced by the binding of ATP (Johnson, et al., Chem Rev 2001, 101, 2243-70).
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Whereas the PKA apoenzyme exists in an open conformation that binds peptide
substrate poorly, coordination of ATP rotates the large and small enzyme
lobes,
allowing substrate to bind the enzyme in a catalytically active, closed
conformation
(Akamine, et al., J Mol Biol 2003, 327, 159-71). Our results suggest that 1
recognizes
the open and closed conformations of PKA with similar affinities or,
alternatively, that
the binding of 1 inhibits the conformational changes associated with ATP
binding.
The miniature protein conjugate 1-I~252a was designed after examination of
the ternary complex of PKA with PKIS_z4 and the related indolocarbazole
natural
product staurosporine (Prade, et al., Structure 1997, 5, 1627-37). This
analysis
suggested that an octamethylene chain would appropriately link a C3' amide
derivative
of K252a to the side chain of residue 40 within 1. I~252a analogs with
conservative
substitutions at C3' retain potency against a range of kinases, suggesting
that an
octamethylene chain at this position would be tolerated. Moreover, the PI~AA-
PKIS_24
structure shows the side chain of the corresponding residue of PKIS_24, A21,
pointing
directly into the ATP/staurosporine binding pocket. Accordingly, we
synthesized
chloroacetamide K252a0 (Fi'gure 5) and a derivative of 1 with a cysteine
residue in
place of alanine at position 40 (Figure 6). lA4oc was alkylated with K252 a~
in the
presence of NaI, yielding 1-K252a. K252 a~ was also used to alkylate PI~I'~lc
to
produce PI~I-K252a.
The inhibitory potencies of 1, 1-K252a, PKI-K252a, and K252a itself were
measured using an assay based on streptavidin-matrix capture of biotinylated,
~32P]-
phosphorylated substrates in which ATP and peptide substrate concentrations
were
fixed below their respective KM values. As expected, K252a was a potent PISA
inhibitor (ICso = 0.140 ~ 0.003 nM) (Figure 7c) and the potency of 1 was
similar to its
PKA affinity (ICso = 117 ~ 14 nM) (Figure 7d). The miniature protein conjugate
1-
K252a was 30-fold more potent (ICso = 3.65 ~ 0.13 nM) than 1 alone (Figure
7e).
Interestingly, the analogous molecule PI~I-K252a was 60-fold less potent (ICso
= 221 ~
2 nM) than 1-K252a (Figure 7f) and far less potent than PKI (KI = 2.3 nM)
(Cheng, et
al., J Biol Chem 1986, 261, 989-92). Both 1-K252a and PKI-K252a were far more
potent than variants of lA4oc or PKI'°'mc alkylated with bromoacetamide
in place of
I~252 a0 (ICSO > 1 ~,M, data not shown). The differential potencies of 1-
I~252a and
PI~I-K252a may arise from differences in the affinity of l and PKIS_24 for the
unique
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conformation of PKA observed in ternary complex with PKIS_z4 and
staurosporine.
To evaluate the extent to which 1 alters the kinase specificity of K252a, the
phosphotransferase assay described above was reconfigured to assay the
activities of
four distinct but related protein kinases. Akt kinase (PKB), protein kinase
Coc (PI~C-oc,)
Ca++/calmodulin kinase II (Ca zKII), and cGMP-dependent protein kinase (PKG)
are
all inhibited by K252a (Figure 7c) but not by PKIS_24. Both 1 and 1-K252a
showed
remarkable specificity for PKA, inhibiting no other kinase tested at
concentrations as
high as 100 nM (1-K252a) or 5 ~,M (1) (Figure 7d-e). The only other kinase
inhibited
by 1-K252a was PKG (ICSO = 679 ~ 202 nM), the kinase most similar to PKA
(Glass, et
al., J Biol Chem 1986, 261, 12166-71). By contrast, PKI-K252a displayed low
specificity, inhibiting all kinases tested with ICSO values within a 4-fold
range (Figure
7f). In summary, the PKIS_24 conjugate PKI-K252a displayed lower potency than
K252a and lower specificity than PKIS_2~whereas the miniature protein
conjugate 1-
K252a displayed higher specificity than K252a and higher potency than 1. Our
results
suggest that molecules such as 1-K252a that embody elements of protein surface
recognition could represent a viable general strategy for selective kinase
inhibition.
Example 19 - Miniature proteins for activating transcription through
interactions with
the co-activator protein CREB-binding protein (CBP): high affinity ligands for
the CBP
KIX domain.
The complex between the KIX domain of the transcriptional coactivator protein
CBP and the kinase-inducible activation domain (KID) of the transcription
factor
CREB, though also mediated by an ot,-helix, is strikingly different from the
complexes
formed by Bcl-2 family members. The KID-binding groove of the CBP KIX domain
is
quite shallow and more closely resembles the solvent-exposed protein surface
than a
typical oc-helix-binding groove (Radhakrishnan, et al., Cell 1997, 91, 741-
752). In fact,
only one hydrophobic residue of CREB KID is completely buried from solvent in
the
KID~KIX complex, and formation of a high affinity KID~KIX complex requires the
enthalpic driving force provided by phosphorylation of CREB KID on Serl33
(Mestas,
et al., Nat Struct Biol 1999, 6, 613-614; Zor, et al., J Biol Chem 2002, 277,
42241-
42248). Thus, CBP KIX represents a difficult target for molecular recognition,
and
indeed, no small molecule ligands for CBP KIX have been reported. In this
study,
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protein grafting and molecular evolution by phage display are used to identify
phosphorylated peptide ligands that recognize the hydrophobic surface of CBP
KIX
with high nanomolar to low micromolar affinity and high specificity.
Furthermore,
grafting of the CBP KIX-binding epitope of CREB KID onto the aPP scaffold
yields
molecules capable of high affinity and specific recognition of CBP KIX even in
the
absence of phosphorylation.
A. Library design and generation.
The design of a CBP KIX-binding miniature protein (PPKID) library was based
on the alignment of the oc,-helix of aPP and helix B of the CREB KID domain
shown in
Figure 8B. The otherwise unstructured phosphorylated CREB KID (KIDP) domain
forms two a-helices, A and B, when bound to the CBP KIX domain; each helix
contacts a different region of the CBP KIX surface (Radhakrishnan, et al.,
Cell 1997,
91, 741-752). Mutagenesis studies have determined that most (though not all)
of the
residues that comprise the CBP KIX-binding epitope of CREB KIDP are located in
helix B (Radhakrishnan, et al., Cell 1997, 91, 741-752; Parker, et al., Mol
Cell 1998, 2,
353-359), and only residues from helix B were included in the miniature
protein
library. Four hydrophobic residues from CREB KID (Tyr134, I1e137, Leu138,
Leu141) contribute significantly to the free energy of KIDP~KIX complex
formation.
The PPKID library contained three of these four residues (I1e137, Leu138,
Leu141),
and a conservative mutation of the fourth from Tyr to Phe, which in the
context of
CREB KIDP has no effect on CBP KIX binding (Du, et al., Mol Cell Biol 2000,
20,
4320-4327). This mutation was included, along with the complete recognition
site for
protein kinase A (PKA; Arg130, Arg131, Ser133), to promote phosphorylation of
the
miniature protein library ih vitro, if so desired. In the context of CREB
KIDP, the Tyr
to Phe mutation lowers five-fold the Km for phosphorylation by PKA (Du, et
al., Mol
Cell Biol 2000, 20, 4320-4327). The structural scaffold of the a-helical
portion of the
library was provided by six of eight residues (Va114, Leul7, Phe20, Leu24,
Tyr27,
Leu28) from the aPP a-helix that contribute to the hydrophobic core (Glover,
et al.,
Biopolymers 1983, 22, 293-304). Based on our success using a similar approach
to
improve DNA-binding miniature proteins (Chin, et al., J Am Chem Soc 2001, 123,
2929-2930), the five residues from the polyproline helix of aPP known to
participate in
hydrophobic core formation (Pro2, Gln4, Pros, Tyr7, ProB) were varied to all
20 amino
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acids. Our expectation was that the CBP KIX-binding epitope on the oc-helix
would
guide all library members to the CBP KIX surface, and the functional selection
would
identify those library members with increased CBP KIX affinity derived from
packing
of the polyproline helix against the otherwise exposed face of the bound oc-
helix. A 5 x
107-member library of miniature proteins (PPKID Library 1) based on this
design was
generated for use in phage display selection experiments.
B. Selection of phosphorylated miniature protein ligands for CBP KIX.
Initially, eight rounds of selection were performed (selection 1). Each round
included a PKA-catalyzed in vitro phosphorylation step designed to increase
the CBP
KIX-binding affinities of all library members. Phosphorylation of CREB KID is
critical for high affinity recognition of CBP KIX; measurements of the
contribution of
the Ser133 phosphate moiety to the free energy of the KIDP~I~IX complex range
between 1.5 and 3.0 kcal~mol-1 (Mestas, et al., Nat Struct Biol 1999, 6, 613-
614; Zor, et
al., J Biol Chem 2002, 277, 42241-42248). In this selection, GST-KIX was
immobilized on glutathione-coated microtiter plates, and stringency was
increased over
the course of the selection by increasing the binding and washing temperature,
from 4
°C in round 1 to 25 °C by round 3, and by increasing the length
and number of washes,
from 10 x 1 min washes in round 1 to 20 x 5 min washes in round 8. Rounds 7
and 8
were performed in binding buffer containing 5 mM dithiothreitol (DTT), after
sequencing of individual clones from rounds 4-6 indicated that a significant
portion of
the library members selected in these rounds contained single Cys residues.
The Cys
residues were evenly distributed over all five randomized positions, which
suggested
that library members were being selected based on their ability to form
disulfide bonds
with GST-KIX or glutathione, rather than based on high affinity, yet
noncovalent, CBP
KIX binding.
The progress of the selection was monitored by measuring the retention of
library phage in comparison to the retention of phage displaying aPP, which
should not
bind to GST-KIX, and by sequencing of individual clones after each round of
selection.
By round 8 of selection 1, the library phage were retained 13-fold over aPP
phage.
Furthermore, by round 7, three sequences (PPKID 1-3) had been identified in
multiple
independent clones (Table 1); two of these sequences (PPKID2, PPI~ID3)
completely
dominated the library by round 8. Surprisingly, the residues selected at each
of the five
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randomized positions in PPKID2 and PPI~ID3 displayed no significant similarity
(PPKID2 and PPKID3 each contain a spurious mutation not encoded in the
original
library pool, but the mutation is different in each peptide (Tyr to Asp at
position 21 for
PPKID2, Leu to Arg at position 24 for PPKID3)).
Table 1. HisKIX-binding affinity of PPI~ID and control peptidesa
Selection 1 Kd PPK)DP K~ PPK)Du
(nM) (p,M)
PKID1 GASDMTYWGDDAPVRRLSFFYILLDLYLDAPGVC591 59 24.1 4.0
PK)D2 GMSRVTPGGDDAPVRRLSFFYILRDLYLDAPGVC729 36 12.6 1.4
PK)D3 GASPHTSSGDDAPVRRLSFFDILLDLYLDAPGVC1200 100 6.7 -!-
0.2
Selections 2 & 4 Kd ppK)DP Kd ppKIIO
(nM) (~uM)
pK)D4 GPSQPTYPGDDAPVRRLSFFYILLDLYLDAPGVC515 44 12.1 a-
2.4
PKIDS GLSWPTYHGDDAPVRRLSFFYILLDLYLDAPGVC534 31 6.6 -!-
2.0
Selections 3 & 4 Kd PPK)DP Kd PPK)Du
i (nM) (~,M)
PKlD6 GISWPTFEGDDAPVRRLSFFYILLDLYLDAPGVC624 49 1.5 0.1
PK)D6 S18E GISWPTFEGDDAPVRRLEFFYILLDLYLDAPGVC 10.9 -!-
2.0
PKlD7 GLSPYTEWGDDAPVRRLSFFYILLDLYLDAPGVC 2.3 0.2
PKID8 GLSWKTDPGDDAPVRRLSFFYILLDLYLDAPGVC 3.1 0.5
Control peptides P: Kd U: Kd (1tM)
K)D-AB TDSQKRREILSRRPSYRKILNDLSSDAPGVC 562 41 >116
nM
~-$ RRPSYRKILNDLSSDAPGVC 51.6 4.0 >297
SAM
eptide C RRLSFFYILLDLYLDAPGVC 2.4 0.2 21.5 2.6
~,M
a. Each peptide was labeled on the C-terminal Cys residue with
acetamidofluorescein for use in fluorescence polarization experiments. Kd
values were
determined by converting polarization data from three independent samples to
fraction
of fluorescently-labeled peptide bound values, which were fit to equilibrium
binding
equation (2). Residues selected at randomized positions are in red. Selected
point
mutations in PPI~ID2 and PPKID3 are underlined. P indicates a phosphopeptide.
U
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indicates an unphosphorylated peptide. The phosphoserine residue in
phosphopeptides
is in bold.
C. CBP KIX-binding affinity.
The PPKID peptides were synthesized as phosphopeptides (PPKIDP) and each
was labeled with acetamidofluorescein on a C-terminal Cys residue. The
affinity of
each labeled peptide for a His-tagged CBP KIX domain (HisKIX) was measured by
equilibrium fluorescence polarization. The HisKIX-binding affinities of three
phosphorylated control peptides (KID-ABP, KID-BP and peptide CP) were also
measured. Peptide KID-ABP comprises the full-length CREB KID domain (residues
119-148, A and B helices) and peptide KID-BP corresponds to the region of CREB
K117
whose residues were incorporated within the a-helix of aPP (residues 130-148,
the
PKA recognition site and helix B); these peptides allow direct comparison of
our
miniature proteins with natural CBP KIX-binding molecules. Peptide CP
corresponds
to the chimeric oc-helical portion of the PPKID peptides (residues 15-33) and
allows us
to compare the contribution to CBP~KIX-binding affinity of residues in the oc-
helix
derived from aPP and residues in the randomized region of the PPKID library,
which
includes the putative polyproline helix and turn regions.
The results of the equilibrium fluorescence polarization experiments are shown
in Figure 9A and Table 1. Km-ABP binds HisKIX with high affinity (Kd = 562 ~
41
nM) at 25 °C under the assay conditions used. This value is lower than
previously
reported Kds for similar KIDP~KIX complexes (3.1 ~,M to 9.7 ~,M) measured by a
number of techniques (though not fluorescence polarization) and may result
from slight
differences in the buffers and the CREB KIDP and CBP KIX constructs used in
each
case. Peptides PPKIDP 1-3 bind HisKIX with affinities ranging from 591 nM to
1.2
~.M, values that are comparable to the HisKIX-binding affinity of KID-ABP.
Remarkably, peptides PPKIDP 1-3 bind HisKIX with 43- to 87-fold higher
affinity than does KID-BP (Kd = 51.6 ~ 4.0 ~.M; this value is comparable to
the Kd of
80 ~,M reported for the KID(129-149)P~KIX complex measured by isothermal
titration
calorimetry). Most of this increase in affinity can be attributed to the aPP-
derived
residues in the oc-helical region of the miniature proteins; peptide CP (which
comprises
the oc-helical region of PPKIDP 1-3) binds HisKIX with a Kd of 2.4 ~ 0.2 ~.M,
which
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represents a greater than 20-fold increase in affinity (~G = -1.8 kcal~mol-1)
compared
to the CBP KIX-binding affinity of KID-BP. The turn and polyproline helix
regions
(including selected residues) of the PPKIDP 1-3 peptides contribute a more
modest -0.4
to -0.8 kcal~mol-1 to the free energy of complex formation with CBP KIX.
The HisKIX-binding affinities of unphosphorylated versions (denoted by a
superscript U) of PPKID 1-3, KID-AB, KID-B and peptide C were also determined
(Figure 9B and Table 1). As expected, the KID-ABU and KID-BU peptides possess
very low affinities for HisKIX. Only a small change in polarization of the KID-
ABU-
Flu (61 mP) or K~-BU-Flu (76 mP) molecules was observed even at the highest
HisKIX concentrations tested (150 ~,M and 325 ~,M, respectively). This
experiment
allows us to place a lower limit on the Kd of the complex formed between each
of these
peptides and HisKIX. If we estimate the change in polarization of KID-ABU-Flu
to be
110 mP and the change in polarization of KID-BU-Flu to be 150 mP when fully
bound
by HisKIX (based on observed changes in polarization of 116 mP for fully
HisKIX-
bound KID-ABP and 161 mP for KID-BP), we can estimate that the Kd of the KID-
ABU~HisKIX complex must be greater than 116 ~,M and the Kd of the KID-
BU~HisKIX
complex must be greater than 297 ~,M. Remarkably, the seven amino acid changes
(including the conservative Tyr to Phe mutation) that convert KID-BU to
peptide CU
dramatically enhance CBP KIX-binding affinity (~G > -1.5 kcal~mol-1). Peptide
CU
binds HisKIX with a Kd of 21.5 ~ 2.6 ~.M. Addition of the turn and selected
polyproline helix regions to yield peptides PPKIDU 1-3 slightly increases or
even
slightly decreases CBP KIX-binding affinity 1- to 3-fold (Kd = 6.7 to 24.1
~,M; ~G = -
0.7 to +0.1 kcal~mol-1). As is true in the context of phosphorylated peptides,
then, most
of the free energy of complex formation with HisKIX is due to aPP-derived
residues in
the putative a-helical region of the PPKIDU peptides.
D. Minimizing fusion protein binding.
Preliminary fluorescence polarization experiments using GST-KIX as a target
indicated that two of the selected peptides (PPKIDl and PPKID3) possessed
significantly higher (16- to 19-fold) affinity for GST-KIX than for HisKIX
(data not
shown). Therefore, we subjected the members of PPKID Library 1 to a second
selection (selection 2) in which GST-KIX and HisKIX were alternated as the
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immobilized target protein to minimize selection of library members based' on
increased affinity for the GST-KIX or HisKIX fusion proteins relative to the
isolated
CBP KIX domain. Binding and washing conditions were similar to those used in
selection 1, and each round included a PKA-catalyzed phosphorylation step. DTT
(5
mM) was included in the binding buffer in all rounds where GST-KIX was used as
a
target (except for round 1) to minimize selection based on disulfide bond
formation.
After nine rounds of selection, the library phage were retained 44-fold over
phage
displaying aPP, although no consensus in miniature protein sequence was
achieved.
However, two sequences were identified in multiple independent clones from
rounds 7-
9 (PPKID 4-5). Interestingly, PPKID4 contains aPP-derived residues in all
randomized
positions. PPKID4 and PPKTDS contain identical residues at two of the
randomized
positions, 5 (Pro) and 7 (Tyr), but otherwise the selected residues axe not
conserved.
Furthermore, none of the selected residues in either PPK>D4 or PPKID5 are
similar to
the selected residues in PPKID 1-3. PPKID4 and PPKTDS exhibit high affinity
for
HisKIX (Figure 9C and Table 1), with Kas in both phosphorylated (515 ~ 44 nM
and
534 ~ 31 nM, respectively) and unphosphorylated forms (12.1 t 2.4 ~.M and 6.6
~ 2.0
p.M, respectively) similar to those observed for PPKID 1-3.
E. Unphosphorylated selections.
The significant CBP KIX-binding affinity displayed by peptide CU (as well as
by short, unphosphorylated CBP KIX-binding peptides identified by Montminy and
coworkers) encouraged us to perform selections with uhphosphorylated PPKID
Library
1. Unphosphorylated selections (selections 3 & 4) were performed in parallel
with
selections 1 & 2, with similar binding and washing conditions. After nine
rounds of
selection, the library phage in selection 3 were retained 32-fold over phage
displaying
aPP, and the library phage in selection 4 were retained 11-fold over phage
displaying
aPP. Although no consensus was reached in either selection, a number of
sequences
were identified in multiple independent clones. In selection 3, one sequence,
PPKID6,
was identified in rounds 6-9. Two of the sequences identified in selection 4,
PPKID4
and PPKID5, were also identified in selection 2 (which included the
phosphorylation
step in each round) under the same conditions. Two additional sequences,
PPKID7 and
PPKID8, were identified in rounds 6-9 in selection 4.
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Interestingly, four of five randomized positions (2, 4, 5, and 7) in peptides
PPKID 4-9 approach consensus; Leu or Ile was selected ,at position 2, Trp at
position 4,
Pro at position 5, and aromatic or negatively charged residues at position 7.
PPK1176U,
PPKID7U and PPKID8U exhibit exceptionally high affinity for HisKIX, as
measured by
fluorescence polarization, with Kds ranging from 1.5 ~,M to 3.1 ~.M (Figure 9C-
D and
Table 1). These values correspond to at least 37- to 77-fold enhancements in
HisKIX-
binding affinity compared to KID-ABU and at least 96- to 198-fold enhancements
relative to KID-BU. Furtherinore, peptides PPKIDU 6-8 bind HisKIX with 7- to
14-fold
enhancements in binding affinity compared to peptide CU. Thus, the selected
polyproline helix and turn regions of the PPKIDU 6-8 peptides contribute -1.2
to -1.6
kcal~mol-1 to the free energy of complex formation with CBP KIX.
We investigated the HisKIX-binding affinities of two variants of PPKID6, each
containing a simple modification of residue Serl8, phosphorylation and
substitution of
Ser by Glu. Phosphorylation of PPKID6 leads to only a two-fold enhancement in
HisKIX-binding affinity (~G = -0.5 kcal~mol-1) (Figure 9D), a significantly
smaller
enhancement than is observed upon phosphorylation for the other PPK~ peptides
(6-
to 41-fold; ~~G = -1.0 to -2.2 kcal~mol-1) and KID-AB (O~G > 3.2 kcal~mol-1).
Surprisingly, the Ser to Glu mutation actually decreases HisKIX-binding
affinity 7-fold
(Kd = 10.9 ~ 2.0 ~,M; ~G = +1.2 kcal~mol-1). A similar mutation in the context
of the
full length CREB KID domain leads to CBP KIX-binding affinity intermediate
between
that of unphosphorylated and phosphorylated CREB KID (Shaywitz, et al.,
Mol~Cel1
Biol 2000, 20, 9409-9422) presumably because the negative charge of Glu mimics
the
negatively charged phosphate moiety.
F. Binding modes of PPK1174P and PPKID6U
Two sets of experiments were performed to investigate the binding modes of
PPKID4P and PPKID6U. First, competition fluorescence polarization experiments
assessed the ability of PPKID4P and PPKID6U to compete with CREB KIDP for
binding CBP KIX. In particular, the fraction of fluorescently tagged PPKID4P
or
PPKID6U bound to HisKIX at equilibrium was monitored as a function of the
concentration of unlabeled KID-ABP. These experiments reveal that Km-ABP
competes with both PPKID4P and PPKID6U for binding to CBP KIX (Figure 10). The
concentration of K~-ABP needed to displace 50°l0 of fluorescently
tagged PPKID4P or
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pPKID6U from HisKIX (the ICso value) is 3.2 ~,M or 2.4 ~.M, respectively.
These
values are, as expected given the conditions of the assay (Munson, et al., J
Recept Res
1988, 8, 533-546), slightly larger than the Kd of the KID-ABP~HisKIX complex
determined by direct fluorescence polarization analysis (562 ~ 41 nM). These
results
indicate that HisKIX cannot interact simultaneously with KID-ABP and either
PPKID4P
or PPKID6U, and axe consistent with an interaction of both PPKID4P and PPK~6U
within the CREB KIDP-binding cleft of CBP KIX.
Although KID-ABP competes with both PPKID4P and PPKID6U for binding to
HisKIX, small changes in KID-ABP concentration axound the corresponding ICso
values have a larger effect on the change in the fraction of PPKID4P bound
than on the
change in the fraction of PPKID6U bound. This result suggests that there may
exist
differences in the orientation or geometry of PPKID4P and PPKID6U when bound
to
CBP KIX. To explore these differences in greater detail, we measured the
affinities of
KID-ABP, PPKID4P and PPKID6U for the Y650A variant of CBP KIX (GST-KIXY6soa)
using direct fluorescence polarization analysis (Figure 11). Tyr650 forms one
side of
the hydrophobic cleft within the CREB KIDP-binding groove of CBP KIX that
accommodates Leul41 of helix B. As a result, CREB K117P exhibits significantly
lower affinity for the Y650A variant relative to wild type CBP KIX. These two
factors
make GST-KIXy6soa an excellent surveyor of the CREB KIDP~CBP KIX interface.
The GST-KIXygsoA~K~-ABP Complex is 15-fold less stable than the wild type
GST-KIX~KID-ABP complex as measured by fluorescence polarization, a difference
in
stability similar to that observed previously in ITC experiments performed
with the
same GST-KIX constructs. Likewise, the PPKID4P~ GST-KIXY6soa complex is 24-
fold
less stable than the wild type GST-KIX~PPKID4P complex. The observation that
mutation of Tyr650 to Ala has a similar effect on the binding of KID-ABP and
PPKID4P, together with the equilibrium competition analysis, provides evidence
that
the two ligands, interact with CBP KIX in a similar manner. Interestingly,
despite the
fact that PPKID6U and CREB KID-ABP compete for binding to CBP KIX, PPKID6U
binds GST-KIXY6soa with the same affinity (Kd = 712 ~ 68 nM) as it binds wild
type
GST-KIX (Kd = 714 ~ 128 nM). This observation suggests that PPKID6°
interacts
with CBP KIX in a manner that is somewhat different from the CBP KIX-binding
mode of KID-ABP. Further work with an established panel of CBP KIX variants
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(Parker, et al., Mol Cell Biol 1999, 19, 5601-5607) currently in progress,
will be
necessary to characterize the binding mode of PPKID6U in detail.
G. PPKID specificity.
Given the myriad protein surfaces present in the cell, the utility of
molecules
that recognize protein surfaces will depend on their ability to interact
selectively with
the desired protein. We investigated the specificity of our highest affinity
phosphorylated (PPKm4P) and unphosphorylated (PPKID6U) CBP KIX ligands by
measuring their affinity for two globular proteins, carbonic anhydrase II and
calmodulin, known to recognize hydrophobic or helical molecules. To determine
the
effect of the region comprising the selected polyproline helix and turn on the
specificity
of PPK1174P and PPKID6U, we also examined the specificity of peptide C, in
both its
phosphorylated and unphosphorylated forms.
The PPKm peptides bind carbonic anhydrase with low affinity, with Kds of 106
~ 12 ~.M and 79 ~ 13 ~.M for PPKID4P and PPKID6U, respectively (Figure 12A and
Table 2). These values define specificity ratios (Krei = Ka (carbonic
anhydrase) / Kd
(HisKIX)) of 205 for PPKID4P and 53 for PPKID6U. The preference of PPKID4P for
HisKIX over carbonic anhydrase (Krei = 205) is considerably higher than the
preference
of control peptide CP for HisKIX over carbonic anhydrase (Krei = 40), despite
their
approximately equal affinity for carbonic anhydrase (106 pM and 97 pM,
respectively).
Thus, the increased specificity of PPKID4P relative to peptide CP is due to
enhanced
affinity for HisKIX, and not a result of decreased affinity for carbonic
anhydrase.
Similar conclusions are drawn when comparing PPKID6U and peptide CU; although
these two molecules display similar affinities for carbonic anhydrase, with Kd
values of
79 ~M and 66 ~M, respectively, the specificity ratio for PPKID6U (Krei = 53)
is
significantly higher than the specificity ratio for peptide CU (Krel = 3).
Table 2: Specificity of PPKID and control peptidesa
Kd HisKIXKd CA Kd CaIM
(~M)
(~.,t,M) (Kret) (!-~M)
Pe tides (Krel)
PPKID4P GPSQPTYPGDDAPVRRLSFFYILLDLYLDAPGVC0.515 106 52
0.044 12 12
(205) (100)
PPKID6U GISWPTFEGDDAPVRRLSFFYILLDLYLDAPGVC1.5 '!' 79 -!- > 168
0.1 13
(53) (> 112)
C P RRLSFFYILLDLYLDAPGVC 2.4 0.2 97 6 178
42
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(40) (74)
C U RRLSFFYILLDLYLDAPGVC 21.5 66 11 N.D.
2.6
a. Each peptide was labeled on the C-terminal Cys residue with
acetamidofluorescein for use in fluorescence polarization experiments. Kd
values were
determined by converting polarization data from two to three independent
samples to
fraction of fluorescently-labeled peptide bound values, which were fit to
equilibrium
binding equation (2). Residues selected at randomized positions are in red. P
indicates
a phosphopeptide. LT indicates an unphosphorylated peptide. The phosphoserine
residue in phosphopeptides is in bold. CA indicates that carbonic anhydrase II
was
used as the target protein; CaIM indicates calmodulin was used as the target
protein.
The specificity ratio Krel 1S defined as Krei = Ka (CA or CaIM) / Kd (HisKIX).
N.D.
indicates that the value was not determined.
The selected PPKff~ molecules also display a dramatic preference for binding
CBP KIX over calmodulin (Figure 12B and Table 2). PPKID4P binds calmodulin
with
a Kd of 51 ~ 12 ~.M, which corresponds to a Krei value of 100. Peptide CP
displays
slightly lower specificity (Krei = 74) than PPK1174P for CBP KIX over
calmodulin, a
result of 5-fold lower affinity for HisKIX and 4-fold lower affinity for
calmodulin (Kd
= 178 ~ 42 ~,M). .The Kd for the PPKID6U~calmodulin complex could not be
determined definitively, but we could place a lower limit of 168 ~.M on the Kd
value by
defining the minimum change in polarization between the fully calmodulin-bound
and
fully unbound states of fluorescently labeled PPKID6U as 100 mP (the observed
change
in the presence of 185 ~.M calmodulin was 66 mP). Thus, PPKID6", like PPKID4P,
exhibits a significant preference for CBP KIX over calmodulin, with a
specificity ratio
of at least 112.
In sum, the work described here extends the utility of the protein grafting
and
molecular evolution procedure to the significant problem of high affinity and
specific
recognition of shallow protein surfaces. Taken together with previous
applications, the
protein grafting strategy has now proven to be extremely general in scope,
enabling the
discovery of highly functional miniature proteins capable of molecular
recognition of
diverse nucleic acid and protein targets. In addition, a posttranslational
modification
step, phosphorylation, was introduced here for the first time into the
molecular
evolution protocol used in protein grafting. Phosphorylated peptide ligands
based on
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the functional epitope of the CREB KID domain were discovered which possess
high
nanomolar to low micromolar affinity and high specificity for the shallow
surface
groove of the CBP KIX domain. Furthermore, presentation of the CREB KID domain
functional epitope on the aPP scaffold protein yielded peptide ligands for CBP
KIX
which bypass the need for phosphorylation to achieve high affinity CBP KIX
recognition and have potential for use as extremely potent transcriptional
activation
domains.
H. Experimental Section.
1) HisKIX expression vector cloning-- The CBP KIX-coding region (residues
586 to 672) of pGEX-KT KIX 10-672 (a gift from Marc Montminy) (Parker, et al.,
Mol
Cell Biol 1999, 19, 5601-5607) was amplified by PCR using 5' and 3' primers
containing NdeI and BamHI restriction sites, respectively. Primers KIXSP and
KIX3P
had the following sequences: KIX5P: 5'-GCCGCGCGGCA GCCATATGGG TGTTC
GAAAAGCCTGGC-3'; KIX3P: 5'-CCAGGCCGCTGCG GATCCTCATCATAA
ACGTGACCTCCGC-3'. The CBP KIX-coding duplex DNA insert was digested with
NdeI and BamHI and ligated into NdeI- and BamHI-digested pETl5b (Novagen)
using
T4 DNA ligase (New England Biolabs). The resulting plasmid, pHisKIX, codes for
the
CBP KIX domain in-frame with an amino-terminal hexahistidine tag under control
of a
T7 promoter. Plasmid identity was confirmed by DNA sequencing of the CBP KIX-
coding region of pHisKIX.
2) Overexpression and purification of GST-KIX and HisKIX-- pGST
OKIX(588-683) (a gift from Jennifer Nyborg) (Yan, et al., J Mol Biol 1998,
281, 395
400) or pHisKIX was transformed into BL21(DE3) pArg E. coli cells by
electroporation. A single colony was used to inoculate a 1 L culture of LB
media
containing 0.2 mg/mL ampicillin and 0.05 mg/mL kanamycin. The culture was
incubated at 37 °C with shaking at 250 rpm until the solution reached
an optical density
of 0.6 absorbance units at 600 nm. Isopropyl (3-D-thiogalactoside (IPTG) was
added to
a final concentration of 1 mM and incubation continued for 3 h at 37
°C. Cells were
harvested by centrifugation for 20 min at 10,800 g and resuspended in 15-20 mL
of
buffer (GST-KIX: 50 mM potassium phosphate (pH 7.2), 150 mM NaCl, 1 mM DTT;
HisKIX: 50 mM sodium phosphate (pH 8.0), 300 mM NaCI, 10 mM imidazole). Cells
were lysed by sonication, insoluble material was pelleted by centrifugation
for 30 min
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at 37,000 g, and the supernatant was retained. GST-KIX and HisKIX proteins
were
purified by glutathione and nickel-nitrolotriacetic acid (Ni-NTA) affinity
chromatography, respectively. Fractions containing the desired protein were
combined,
desalted on a NAP 10 (GST-KIX) or NAP 25 (HisKIX) column (Amersham) and
stored in buffer containing 50 mM Tris (pH 8.0), 100 mM KCI, 12.5 mM MgCl2, 1
mM
ethylenediaminetetraacetic acid (EDTA) and 0.05% Tween-20 (GST-KIX storage
buffer also contained 1 mM DTT) at -70 °C. Protein identity and
concentration were
confirmed by amino acid analysis.
3) Phage library construction-- PPKID Library 1 was created by cassette
mutagenesis of the phagemid vector pJC20 (Chin, et al., Bioorg Med Chem Lett
2001,
11, 1501-1505) using the synthetic oligonucleotides Alignl and PPLib. These
oligonucleotides possessed the following sequences (N indicates an equimolar
mixture
of G, C, A and T, and S indicates an equimolar mixture of G and C): Alignl: 5'-
TGTTCCTT TCTATGCACCGGTTCGTCTC TGTCC TT
CTTCTACATCCTGCTGGACCTGTACC TGGACGCACCGGCGGC
CGCAGGTGCGCCGGGCC-3'; PPLib: 5'-TGTTCCTTTCTAT GCGGCCCAGCCG
GCCGTNNS TCCNNSNNSACCNNSNNSGG TGACGACGCACCG
GTAGGTGCGCC GGTGCC-3'. Double stranded Alignl and PPLib inserts were
generated by primer extension of appropriate primers using Sequenase version
2.0 T7
DNA polymerase (IJS Biochemicals). The duplex Alignl insert was digested with
AgeI and NotI, and purified from a preparative agarose gel using the QIAquick
gel
extraction kit (Qiagen) and ethanol precipitation. Purified Alignl insert was
ligated
into AgeI- and NotI-digested pJC20 using the Ligation Express Kit (Clontech)
to yield
the phagemid vector pAlignl. Double stranded PPLib insert was digested with
AgeI
and SfiI and purified as per Alignl. PPLib insert was ligated into AgeI- and
SfiI-
digested pAlignl using the Ligation Express Kit (Clontech) to generate PPKID
Library
1. The ligated PPKm Library 1 phagemid vector was transformed into XLl Blue E.
coli cells by electroporation and amplified by overnight growth at 37
°C in 2X YT-AG
media (2X YT media containing 2% (w/v) glucose and 0.1 mg/mL ampicillin).
Glycerol stocks of this culture were used as the initial pool for selection
experiments.
PPKI17 Library 1 contained 5 x 107 independent transformants, which covered
the
theoretical diversity of the library (325 = 3.36 x 107) with 77% confidence.
Sequencing
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of twenty individual clones from the initial pool verified the quality of the
library; none
of the sequenced clones contained mutations, deletions or insertions in the
PPKID-
coding region.
4) Phage display procedure-- A glycerol stock of the initial pool (round 1) or
output from the previous round (rounds 2-9) was used to inoculate 10 mL 2X YT-
AG
media. The culture was incubated at 37 °C until it reached an optical
density of 0.6
absorbance units at 600 nm. The culture was then infected with 4 x 1011 pfu
M13K07
helper phage and incubated at 37 °C for 1 h. Cells were pelleted by
centrifugation,
resuspended in 10 mL 2X YT-AID (2X YT media containing 0.1 mg/mL ampicillin
and
0.05 mg/mL kanamycin) and incubated for 12-13 h at 37 °C. Cells were
then pelleted
by centrifugation and the retained supernatant was filtered through a 0.45 ~.m
syringe
filter. Phage were precipitated with 1/5 volume PEG/NaCI (20% (w/v) PEG-8000,
2.5
M NaCI) on ice for 45 min, and then pelleted by centrifugation for 35 min at
24,000 g.
For phosphorylated selections, the precipitated phage were resuspended in
water and
approximately 101° phage were phosphorylated ifa vitro with 2500 U PKA
(Promega) in
100 ~.M ATP, 40 mM Tris (pH 8), 20 mM magnesium acetate for 2 h at 30
°C.
Phosphorylated phage were precipitated on ice for 45 min with PEG/NaCI and
then
pelleted by centrifugation at maximum speed in a microcentrifuge for 30 min.
Mock
phosphorylation reactions were performed in parallel without PKA, and purified
in the
same manner. Precipitated phage (+/- PISA treatment) were resuspended in
binding
buffer for use in selections. HisI~IX binding buffer contained 50 mM potassium
phosphate (pH 7.2), 150 mM NaCI, 0.05% Tween-20 and GST-I~IX binding buffer
contained 20 mM Tris (pH 8.0), 150 mM NaCI, 0.1 % Tween-20.
Selections against HisI~IX were performed in Ni-NTA HisSorb microtiter 8-
well strips (Qiagen) and selections against GST-I~IX were performed in
glutathione-
coated 96-well microtiter plates (Pierce). 200 ~.L target protein was added to
each well
(final concentration of 30 nM for GST-KIX and 100 nM for HisKIX) and incubated
overnight with shaking at 4 °C. Wells were washed three times with
HisKIX or GST-
KIX binding buffer to remove unbound protein. For blocking, binding buffer
containing 6% milk was added to each well and incubated at 4 °C for 3
h. After
bloclcing, wells were washed three times with binding buffer. Phage purified
as
described were added to each well and incubated for 3 h at 4 °C or 25
°C. Nonbinding
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or weakly binding phage were removed by repeated washing (10 to 20 times, 1
min to 5
min in length, according to round) with binding buffer. Bound phage were
eluted by
incubation with 0.1 M glycine (pH 2.2) for 20 min. After neutralization of the
eluted
phage solution with 2 M Tris (pH 9.2), XLl Blue E. coli cells in log phase
were
infected with input and output phage and incubated at 37 °C for 1 h.
Serial dilutions of
infected cells were plated on SOB agar plates containing 2% (wlv) glucose and
0.1
mg/mL ampicillin. Cells infected with output phage were used to make glycerol
stocks
and stored at -70 °C.
5) Peptide synthesis and modification-- Peptides were synthesized on a 25
~.mol scale at the HHMI Biopolymer/Keck Foundation Biotechnology Resource
Laboratory at the Yale University School of Medicine, New Haven, CT. All
peptides
contained free N-terminal amines and amidated C-termini. Phosphoserine
residues
were introduced using an Fmoc-protected O-benzyl-phosphoserine derivative with
standard coupling conditions. Crude peptides were purified by reverse-phase
HPLC on
a Vydac semi-preparative C18 column (300 A, 5 ~.m, 10 mm x 150 mm). Matrix-
assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass
spectrometry
was used to confirm peptide identity before further modification. Fluorescein-
conjugated derivatives were generated by reaction of purified peptides
containing
single C-terminal cysteine residues with a 10-fold molar excess of 5-
iodoacetamidofluorescein (Molecular Probes) in a 3:2 mixture of
dimethylformamide:
phosphate-buffered saline (DMF:PBS). Labeling reactions were incubated with
rotation for 3-16 h at room temperature. Fluorescein-labeled peptides were
purified by
reverse-phase HPLC as described above, and characterized by MALDI-TOF mass
spectrometry and amino acid analysis.
6) Fluorescence polarization-- Fluorescence polarization experiments were
performed with a Photon Technology International QuantaMaster C-60
spectrofluorimeter at 25 °C in a 1 cm pathlength Hellma cuvette. Serial
dilutions of
HisKIX were made in buffer containing 50 mM Tris (pH 8.0), 100 mM KCI, 12.5 mM
MgCl2, 1 mM EDTA, 0.1°Io Tween-20. Briefly, an aliquot of fluorescently
labeled
peptide was added to a final concentration of 25-50 nM and the binding
reaction was
incubated for 30 min at 25 °C. Thirty minutes was a sufficient length
of time for the
binding reaction to reach equilibrium, as judged by an absence of change in
the
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observed polarization value of the sample with the highest HisKIX
concentration over
1 h. For competition experiments, serial dilutions of KID-ABP were incubated
with 1.5
~.M or 3 ~.M HisKIX and 25 nM fluorescein-labeled PPKID4P or PPKID6U
(peptides°)
for 60 min at 25 °C, respectively. For specificity measurements,
carbonic anhydrase II
(Sigma) or calmodulin (Sigma) was used as the target protein in place of
HisKIX, and
fluorescently labeled peptide was used at a final concentration of 50 nM.
Carbonic
anhydrase was serially diluted in binding buffer as described for HisKIX.
Calmodulin
was serially diluted in calmodulin folding buffer containing 20 mM Hepes (pH
7.5),
130 mM KCI, 1 mM CaCl2, 0.05% Tween-20.
Polarization was measured by excitation with vertically polarized light at a
wavelength of 492 nm (10 nm slit width) and subsequent measurement of the
fluorescence emission at a wavelength of 515 nm (10 nm slit width) for 10 s in
the
vertical and horizontal directions. The polarization data were fit using
Kaleidagraph
v3.51 software to equilibrium binding equation (1), derived from first
principles.
(1) P°bs = Pmn + ((PmaX -
P~)~(2[peptideFl°]))([peptideFi°] + [target protein] +
Kd - (([peptides°] + [target protein] + Kd)2 - 4[peptides°]
[target protein])°~s)
In this equation, P°bs is the observed polarization at any target
protein (HisKIX,
carbonic anhydrase or calmodulin) concentration, Pl"~ is the maximum
polarization
value, Pn,;n is the minimum observed polarization value, and Kd is the
equilibrium
dissociation constant. Measurements from two to three independent sets of
samples
were averaged for each dissociation constant determination. For plots of
fraction of
fluorescently labeled peptide (peptideFU) bound as a function of target
protein
concentration, polarization values were converted to fraction of
peptideFl° bound using
the P,r,;n and Pm~ values derived from equation (1), and the fraction of
peptideFU bound
data were fit to equilibrium binding equation (2) using Kaleidagraph v3.51
software.
(2) Gobs = ((1/(2[peptideFl"]))([peptideFl°] + [target protein] + Kd -
(([peptideFi°]
+ [target protein] + Kd)2 - 4[peptides°][target protein])°~5)
In this equation, Robs is the observed fraction of peptideFl° bound at
any target
protein concentration and Kd is the equilibrium dissociation constant.
For competition experiments, observed polarization values were converted to
fraction of peptideFi° bound using experimentally determined Pm;n and
Pm~ values
corresponding to the polarization of samples containing 25 nM
peptideFl° alone and
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peptideFlu with 1.5 ~,M or 3.0 ~.M HisKIX, respectively. The fraction of
peptide~°
bound data were fit to equation (3) using Kaleidagraph v3.51 software to
determine the
ICSO value.
(3) Gobs = ((0max- ~,n;I,)~(1 + ([competitor]/ICS~)S'ope)) + 6~,;I,
In this equation, Bobs is the observed fraction of peptide~u bound at any
competitor peptide concentration, slope is defined as the slope at the
inflection point
and ICSO is the concentration of competitor that reduces binding of peptideFU
by 50%.
Example 20 - Characterization of miniature broteins as high affinity li~ands
for the
CBP KIX domain.
A. Do PPKID4P and PPKID6U occupy the CREB KID site on CBP KIX?
In our previous work, we reported that both PPKID4P and PPKID6U bind wild
type CBP KIX with high affinity. In the case of PPKID4P, the affinity (515 ~
44 nM)
was comparable to that of the full length phosphorylated CREB KID domain (KID-
ABP, Kd = 562 ~ 41 nM); PPKID6U bound CBP KIX approximately three-fold worse
(Kd = 1.5 ~ 0.1 ~M) that did KID-ABP (Rutledge, et al., J Am Chem Soc 2003,
125,
14336-14347). Here we made use of a set of twelve well-studied CBP KIX
variants
(Parker, et al., Mol Cell 1998, 2, 353-359) to compare the binding location
and
orientation of PPKID4P and PPKID6U to that of KID-ABP, whose interactions with
CBP KIX have been characterized in detail by NMR (Radhakrishnan, et al., Cell
1997,
91, 741-752). These twelve CBP KIX variants each contain a single alanine
substitution within or around the CREB-binding groove, and their affinities
for KID-
ABP span a 3.7 kcal~mol-1 range (Table 3). We reasoned that if PPKID4P and
PPKID6U
interact with CBP in a manner that mimics that of KID-ABP, their affinities
for these
twelve variants should parallel those of KID-ABP. The relative affinities of
KID-ABP,
PPKID4P and PPKID6U for the panel of CBP KIX variants were measured by
equilibrium fluorescence polarization. The results of these experiments are
shown in
Figure 13 and Table 3.
B. Comparison of the binding modes of PPKID4P and KID-ABP.
~ Recogtiitiofi of phosphoserif2e. Recognition of the phosphoserine residue in
KID-ABP by CBP KIX contributes heavily to the stability of the complex; loss
of the
phosphate results in a 3.5 kcal~mol-1 loss in binding free energy. Therefore
we first
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examined whether the PPK1D4P phosphoserine contributes significantly to the
binding
energy of the PPKID4P~CBP KIX complex. The CBP KIX variants Y658F and K662A
each contain a mutation of a residue that directly contacts the KID-ABP
phosphoserine
(Figure 14A). The Y658 side chain donates a phenolic hydrogen bond to one
terminal
phosphoserine oxygen whereas the K662 ammonium group forms a salt bridge with
a
second terminal phosphoserine oxygen. The Y658F and K662A CBP KI:X variants
both exhibit significantly decreased affinity for KID-ABP, consistent with
previous
results, with equilibrium dissociation constants of 26 ~ 5 and 4.8 ~ 0.4 ~.M,
respectively. These values correspond to binding free energies that are 2.5
and 1.5
kcal~mol-1 less favorable, respectively, than the wild type KID-ABP~CBP KIX
complex. The free energy changes we measure with these two variants, as well
as the
Y658A variant (discussed below), are consistent with previous work and
available
structural information.
The Y658F and K662A variants of CBP KIX also exhibit significantly
decreased affinity for PPKID4P. The equilibrium dissociation constant of the
PPKID4P~Y658F complex is 4.1 ~ 0.2 ~,M. This value corresponds to a binding
free
energy that is 1.1 kcal~mol-1 less favorable than that of the wild type
PPKID4P~CBP
KIX complex, approximately one half the magnitude of the change seen with KID-
ABP. The equilibrium dissociation constant of the K662A~PPKID4P complex is 3.9
~
0.3 ~.M. This value corresponds to a binding free energy that is 1.1 kcal~mol-
1 less
favorable than that of the wild type PPKID4P~CBP KIX complex, exactly the
value
measured with KID-ABP. These data suggest that the Y658 hydrogen bond and the
K662 salt bridge each contribute significantly to the affinity of PPKID4P for
CBP KIX.
Interestingly, the hydrogen bond to Y658 is more important overall for KID-ABP
than
for PPKID4P, whereas the salt bridge with K662 contributes almost equally for
both
peptides.
We also examined the affinities of KID-ABP and PPKID4P for the Y658A
variant of CBP KIX in which the entire tyrosine side chain has been replaced
by
alanine. The NMR structure of the KID-ABP~CBP KIX complex shows this aromatic
ring packed against residue L128 on helix A of KID-ABP. This variant binds KID-
ABP
with exceptionally low affinity, (Kd = 142 ~ 12 ~.M) a loss in binding free
energy of 3.5
kcal~mol-1, presumably because the complex suffers from loss of both the
phenolic
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hydrogen bond and an important hydrophobic packing interaction. Since PPKID4P
lacks a residue corresponding to L128 on helix A, one would expect the
stability of the
PPKID4P~Y658A complex to be comparable to that of the PPKID4P~Y658F complex.
Indeed, the equilibrium dissociation constant of the PPKID4P~Y658A complex is
4.1 ~
0.2 ~.M, corresponding to a free energy loss of 1.1 kcal~mol'1, a value
identical to that
measured for the Y658F~PPKID4P complex.
Hydrophobic contacts. Next we considered those residues that line the shallow
KID-ABP binding cleft on CBP KIX. Six of the twelve variants (L599A, L603A,
K606A, Y650A, LL652-3AA and I657A) contain alanine in place of a residue
within
this cleft. For example, Y650 of CBP KIX comprises one face of the binding
cleft and
interacts with three hydrophobic side chains of KID-ABP, including L138, L141
and
A145 on KID-ABP (Figure 14B). The Y650A variant binds KID-ASP with
significantly
reduced affinity (Kd = 9.4 ~ 0.7 ~M), corresponding to a 1.8 kcal~mol-1 loss
in binding
energy. Together, residues L603 and K606 form one side of the binding cleft of
CBP
KIX, interacting with CREB residues L141 and A145. The L603A and K606A
variants
bind KID-ABP with equilibrium dissociation constants of 3.4 ~ 0.3 and 2.3 ~
0.2 ~t,M,
corresponding to losses in binding free energy of 1.2 and 1.0 kcal~mo1-1
compared to
wild type. Other CBP KIX residues that comprise part of the hydrophobic cleft
are
L653 and I657; both interact with CREB residue L141 in addition to other
residues. As
expected, variants LL652-3AA and I657A also bind KID-ABP with lower affinity
(Kd =
2.9 ~ 0.2 and 1.9 ~ 0.1 ~M), corresponding to losses in binding free energy of
1.2 and
0.9 kcal~mol-1, respectively. Located at the bottom of the binding cleft,
residue L599
interacts with only one residue, P146, of CREB. The L599A variant binds KID-
ABP
with lower affinity, but to a lesser extent than other variants that make up
the
hydrophobic cleft; the KID-ABP~L599A complex has an equilibrium dissociation
constant of 1.1 t 0.1 ~u,M, corresponding to a loss in binding free energy of
0.58
kcal~mol-1. Thus, as expected, all CBP KIX variants of residues known to make
hydrophobic contacts with CREB bind KID-ABP worse than wild type CBP KIX.
All six CBP KIK variants within the hydrophobic binding cleft also show
diminished affinity for PPKff~4P, with equilibrium dissociation constants
between 1.6 ~
0.1 and 3.4 ~ 0.5 ~M. These Kd values correspond to the free energy changes
between
0.58 and 1.0 kcal~mol-1. Amazingly, the magnitude and rank order of the
affinities of
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these six CBP KIX variants for PPKID4P mirror, with ony one exception, the
affinities
measured for KID-ABP. The sole exception to this trend is CBP KIX variant
Y650A;
this variant forms a complex with KID-ABP that is 1.8 kcal~mol-1 less stable
than the
wild type complex whereas the complex with PPKID4P is only 0.95 kcal~mol-1
less
stable.
Residues surrounding the binding pocket. In addition to the variants described
above which contain mutations within the KID-ABP binding pocket, we also
examined
three variants - E655A, I660A and Q661A - with an alanine substituted at a
position
surrounding the binding pocket. These variants may provide additional
information
about the binding site of ligands that do not bind to CBP KIX in the exact
same
orientation as KID-ABP. The equilibrium dissociation constants of the
complexes of
these variants with KID-ABP fall between 0.27 ~ 0.03 and 0.93 ~ 0.07 pM,
values very
close to that of the wild type complex (DOG = -0.26 and +0.48 kcal~mol-1,
respectively). These three CBP KIX variants bind PPKID4P with equilibrium
dissociation constants between 0.54 ~ 0.05 and 1.1 ~ 0.1 pM, corresponding to
free
energy changes between -0.07 and 0.35 kcal~mol-1; similar to KID-ABP, these
values
axe also close to those of the wild type complex.
Table 3. Equilibrium dissociation constants of complexes between PPKID4P,
PPKID6U
and KID-ABP and selected CBP KIX variants.
Ka (!~M)(kcal Kd (N~M) (kca~mol~l)~d (I~) ~G i
0l-1) (kcal
BP I~ mol-
)
Km-ABP PFK)D4P PPI~6U
wild 0.41 0.61 0.54
type 0.04 0.04 0.06
Phosphoserine
contacts
Y658A 14212 3.5 3.90.3 1.1 1.70.4 0.68
Y658F 26 5 2.5 4.1 ~- 1.1 2.8 0.97
0.2 0.4
K662A 4.80.4 1.5 3.90.3 1.1 1.80.3 0.71
Hydrophobic
contacts
within
cleft
Y650A 9.4 1.8 3.0 0.3 0.95 1.3 0.52
0.7 0.2
L603A 3.40.3 1.2 3.40.5 1.0 2.50.3 0.91
LL652- 2.9 ~- 1.2 2.9 0.2 0.93 0.14 -0.80
3AA 0.2 0.02
K606A 2.3 1.0 3.1 0.2 0.97 1.2 ~- 0.47
0.2 0.1
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I657A 1.90.1 0.90 2.70.1 0.89 3.60.4 1.1
L599A 1.10.1 0.58 1.60.1 0.58 3.30.3 1.1
Hydrophobic
contacts
outside
cleft
Q661A 0.93 0.070.48 1.1 0.1 0.35 0.41 0.06 -0.16
E655A 0.71 0.050.32 0.54 0.05-0.07 1.7 0.2 0.68
I660A 0.27 0.03-0.26 1.1 0.1 0.35 6.1 0.7 1.4
C. Comparison of the binding modes of PPKID6U and KID-ABP.
Recognition ofphosplaose~ihe. PPKID6U lacks the phosphoserine that
dominates the energetics of the PPKID4P~CBP KIX and KID-ABP~CBP KIX
complexes. Therefore, if this ligand is bound in a manner that mirrors that of
PPKID4P
and KID-ABP, it should be less sensitive to changes at positions Y658 and K662
of
CBP KIX. Surprisingly, PPKID6U binds both variants with significantly
decreased
affinity relative to wild type CBP KIX. The equilibrium dissociation constants
of the
Y658F~PPKID6U and K662A~PPKID6U complexes are 2.8 ~ 0.4 and 1.8 ~ 0.3 ~,M,
corresponding to free energy losses of 0.97 and 0.71 kcal~mol-I, respectively,
relative to
the wild type complex. Interestingly, the Y658A variant binds PPKID6U better
(K~
1.7 ~ 0.4 ~,M) than the Y658F variant (I~ = 2.8 ~ 0.4 ~.M), whereas Y658A and
Y658F
bind PPKID4P equally well. These results are not consistent with a model in
which the
a-helix in PPKID6U is positioned within the CBP KIX cleft in a manner that
closely
mimics that of KID-ABP. In contrast, they support a model characterized by an
overlapping, but alternate, binding mode for PPKID6U compared to PPKID4P and
KID-
ABP.
Hydrophobic contacts. Mutation of the KIX side chains that line the KID-ABP
binding pocket in CBP KIX (variants L599A, L603A, K606A, Y650A and I657A)
results in complexes with PPKID6° that are 0.47 to 1.1 kcal~mol-1 less
stable than the
wild type complex. In contrast, the complex with LL652-3AA is 0.8 kcal~mol-1
more
stable. Variants L599A, L603A, and I657A exhibit moderately diminished binding
affinity to PPKID6U (Kd = 3.3 ~ 0.3, 2.5 ~ 0.3, and 3.6 ~ 0.4 ~tM; ~G = 1.1,
0.91, and
1.1 kcal~mol-1, respectively), whereas K606A and Y650A show smaller decreases
in
PPKID6U binding affinity (Kd =1.2 ~ 0.1 and 1.3 ~ 0.2 ~,M; SAG = 0.47 and 0.52
kcal~mol-1, respectively). Ranking of the hydrophobic contact residues in
order of
energetic contribution to complex formation with each ligand reveals a pattern
for
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PPKID6U binding unlike that for KID-ABP or PPKID4P. For example, Y650 and L599
make the largest and smallest energetic contributions, respectively, to
binding of KID-
ABP, whereas Y650 contributes least and L599 contributes most to complex
formation
with PPI~ID6U.
Residues sut-~ounding the binding pocket. Although CBP KIX variants E655A,
I660A and Q661A display affinities for I~ID-ABP and PPKID4P comparable to wild
type CBP KIX, two of these variants show significantly diminished affinity for
PPKID6U. Variant I660A exhibits the largest decrease in PPKID6U binding
affinity of
all CBP KIX variants in the panel, with an equilibrium dissociation constant
of 6.1 ~
0.7 ~t.M, corresponding to a free energy loss of 1.4 kcal~mol-1. E655A also
exhibits
decreased affinity for PPKID6U, albeit to a lesser extent (Kd =1.7 ~ 0.2 ~.M;
~G =
0.68 kcal~mol-~), whereas Q661A binds PPKID6U with affinity comparable to wild
type
CBP KIX. Again, differences in the relative and absolute contributions of CBP
KIX
residues to the energy of complex formation with PPKID6U compared to PPKID4P
and
KID-ABP suggest an alternate, but overlapping binding site for PPI~.ID6U.
D. Is PPKID4P folded when bound to CBP KIX?
The results described above suggest that the oc-helix in PPKID4P is positioned
within the CBP KIX cleft in a manner that closely mimics that of KID-ABP, but
shed
no light on whether PPKID4P is folded into an aPP-like hairpin conformation
when
bound. We previously reported that PPKID4P displays only nascent a-helicity in
water
in the absence of CBP KIX, as determined by circular dichroism. To explore
whether
PPKID4P folds into an aPP-like hairpin conformation upon binding to CBP KIX,
we
prepared a set of eleven PPKID4P variants in which alanine or sarcosine is
substituted
for a PPKID4P residue that comprises the hydrophobic core in the putative
folded state
(Blundell, et al., Proc Natl Acad Sci U S A 1981, 78, 4175-4179). These
variants
include alanine substitutions along the face of the PPKID4P oc-helix opposite
the face
used to contact CBP KIX (L17A, F20A, L24A, L28A and Y27A) and six variants
with
alanine or sarcosine substitutions along the PPII helix (P2A, P2Z, PSA, PSZ,
P8A and
P8Z). A close-up view of packing in the hydrophobic core is shown in Figure
15.
We hypothesized that if PPKID4P folds upon binding within the CBP KIX
binding cleft into an aPP-like conformation, then the stability of its complex
with CBP
KIX would depend on the presence of residues that comprise the intact
hydrophobic
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core in a manner commensurate with their contribution to core stability, and
the
corresponding variants would display diminished affinity for CBP KIX. However,
if
the N-terminal region of PPI~ID4P interacts with the CBP KIX surface
elsewhere, then
the stability of the PPKID4P~CBP KTX complex would not depend on the
identities of
putative folding residues from the PPI~ID4P a-helix. In this case, the
variants would
display affinity for CBP KIX comparable to wild type PPKID4P.
Table 4. Binding affinities of PPI~ID4P variants for CBP KIX as determined by
fluorescence polarization.
PPKID4P Ka (~M) (kca~mol-1)
Wild type0.61 0.04
Polyproline
helix
variants
P2A 0.87 0.04 0.21
P2Z 0.83 0.08 0.18
P5A 1.02 ~- 0.30
0.09
P5Z 0.80 0.03 0.16
P8A 1.07 0.08 0.33
P8Z 0.78 0.05 0.15
oc-helix
variants
L17A 0.68 0.05 0.07
F20A 2.55 0.25 0.85
L24A 1.16 0.07 0.3 8
Y27A 3.09 0.18 0.96
L28A 0.83 0.11 0.18
E. Effects of PPKID4P variants on CBP I~IX binding.
We used fluorescence polarization analysis to determine the CBP KIX binding
affinities of eight PPI~ID4P variants in which one residue within the aPP
hydrophobic
core had been substituted with alanine. Five of these residues lie on the
internal face of
the aPP helix, whereas three lie on the internal face of the PPII helix. We
also
studied three variants in which a proline residue on the internal face of the
PPII helix
was substituted by sarcosine (Figure 16 and Table 4). The equilibriiun
dissociation
constants of the PPKID4P variant~CBP KIX complexes range from 0.68 ~ 0.05 to
3.09
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~ 0.18 ~.M, corresponding to binding energies between 0.07 and 0.96 kcal~mol-1
less
favorable than the wild type complex. The stabilities of the variant complexes
fall
naturally into three categories. The least stable complexes containing
variants F20A
and Y27A were 0.85 and 0.96 kcal~mol-1 less stable than the wild type complex;
moderately stable complexes containing variants PSA, P8A and L24A were 0.3 to
0.38
kcal~mol-1 less stable than the wild type complex. Six of the variants, P2A,
P2Z, PSZ,
PBZ, L17A and L28A, formed CBP KIX complexes with stabilities that were
similar
(0.07 to 0.21 kcal~mol-i) to the wild type complex. It is striking that that
those side
chains that contribute significantly or moderately to the stability of the
PPKID4P~CBP
KIX complex - F20, L24, Y27, PS and P8 - all lie at the center of the aPP
hydrophobic
core (Figure 15). The aromatic side chain of F20 inserts between the side
chains of
residues P3 and P5, the branched side chain of L24 packs against P8 and F20,
and the
side chain of Y27 packs against P8. By contrast, those side chains that
contribute
minimally to stability - P2, L17 and L28 - lie at the edge of the hydrophobic
core of
aPP and participate in fewer van der Waals interactions. Thus, these results
support a
model in which PPI~ID4p folds (although to a lesser extent than aPP) upon
binding to
CBP KIX into an aPP-like hairpin conformation.
F. Can PPKID peptides function as transcriptional activation domains in
cultured
cells?
2O Eukaryotic transcriptional activators, such as CREB, stimulate gene
expression
primarily by recruitment of the general transcription machinery to the
promoters of the
genes they regulate (Ptashne, et al., Genes & Signals; Cold Spring Harbor
Laboratory:
New York, 2002). These transcription factors are modular in nature, containing
a DNA
binding domain that targets the activators specifically to the gene of
interest, and an
ZS activation domain that binds, and thereby recruits, the transcriptional
machinery. The
functions of these two domains are separable; domains can be swapped among
different
activator proteins, or indeed replaced altogether with non-natural
counterparts, to
obtain molecules with novel activation activities (Ansari, et al., Curr Opin
Chem Bial
2002, 6, 765-772). The development of fully artificial activators is an
important goal in
30 chemical biology, but although there has been considerable success in the
development
of novel DNA binding molecules, the development of artificial activation
domains lags
behind (Mapp, Organic and Biomolecular Chemistry 2003, 1, 2217-2220). By
virtue of
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their ability to bind CBP, a coactivator protein that bridges transcription
factors and the
basal transcription machinery, we hypothesized that PPKID4p and PPKID6~' might
function as artificial activation domains when fused to a heterologous DNA-
binding
domain.
To investigate the activation potential of these ligands, we prepared a series
of
mammalian expression plasmids containing the CBP KIX ligands KID-AB, PPKID4 or
PPKID6 fused to the C-terminus of the Gal4 DNA-binding domain (DBD) (residues
1-
147). As a control, we used pALl, which contained the Gal4 DBD alone. These
constructs were transfected into HEK293 cells along with a previously
described
reporter plasmid containing five Gal4 binding sites upstream of the firefly
luciferase
gene (Figure 17A) and a plasmid encoding Renilla luciferase to control for
variable
transfection efficiency. The cells were incubated at 37 °C for 36 h,
lysed, and the ratio
~ of activity of firefly and Rinella luciferase measured using the Dual-
Luciferase~
Reporter Assay System (Promega). The potency of each activation domain (fold
activation) was determined by dividing the ~ values measured in cells
transfected with
a Ga4 DBD fusion by the ~ value measured in cells transfected with the pAL1
control.
Based on a previous study that found a correlation between the CBP KIX-binding
affinity and activation potency of short peptides (Frangioni, et al., Nat
Biotechnol 2000,
18, 1080-1085), we expected PPKID4P and PPKID6U to activate transcription at
level
comparable to KID-ABP due to their similar affinities for CBP KIX.
G. Activation potential of PPKID4, PPKID6 and KID-AB.
First, we investigated the ability of KID-AB, PPK.ID4, and PPKID6 to activate
transcription in the absence of forskolin, where phosphorylation of cellular
proteins is
not stimulated (Figure 17B). We previously reported that PPKID4 and KID-AB
possess low affinity for CBP KIX under these conditions (Rutledge, et al., J
Am Chem
Soc 2003, 125, 14336-14347) and would not be expected to effectively recruit
CBP to
the Gal4 promoter. Indeed, Gal4 DBD fusion proteins containing PPKID4 or KID-
AB
did not stimulate transcription over basal levels under these conditions. The
fold
increase in transcription measured in cells transfected with plasmids encoding
these
two fusion proteins was no higher than in cells transfected with PPKID6 also
failed to
activate transcription under these conditioins, despite possessing significant
affinity (Kd
=1.5 ~,M) for CBP KIX.
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Next, we investigated the ability of KID-AB, PpKID4 and PPKID6 to activate
transcription under conditions where phosphorylation is stimulated. Addition
of
forskolin to the cell culture media induces the cAMP pathway, which activates
protein
kinase A (PKA) and leads to the phosphorylation of PKA substrates, including
KID-
AB and the PPKID peptides (Gonzalez, et al., Cell 1989, 59, 675-680;
Johannessen, et
al., Cell Signal 2004, 16, 1187-1199). As phosphorylation of KID-AB and PPKID4
dramatically increases their CBP KIX-binding affinity, we expected that
phosphorylation of these ligands will also increase their ability to recruit
CBP and
hence, their transcriptional potency. Indeed, in the presence of forskolin,
KID-AB and
PPKID4 activate transcription 7.5-fold aver basal levels. Phosphorylatian of
PPKID6
also increases its CBP KIX-binding affinity; however, with only 2-fold higher
affinity
for CBP KIX, it was not clear whether PPKID6P would be capable of activating
transcription when PPKID6U could not. In fact, in the presence of forslcolin,
PPKID6
activated transcription 2.5-fold over basal levels.
H. Does transcriptional activation by the PPKID peptides occur via the
CBP/p300
pathway?
Transcriptional activation by CREB KID occurs via recruitment of CBP to
promoters where CREB is bound. However, transcription can be activated by a
variety
of different pathways. Therefore, it was of great interest to investigate
whether, as we
hypothesize, the observed transcription activation potential of PPKID4P and
PPKID6P
is dependent on recruitment of CBP. Towards this end, we compared the
transcription
potential of PPKID4P and PPKID6P in the presence and absence of exogenous
p300, a
paralog of CBP. An increase in the effective concentration of p300 in the
cells should
lead to an increase in transcriptional activation that occurs via the p300/CBP
pathway.
Thus, we expected that the levels of transcription activation elicited by
PPKID4P and
PPKID6P in the presence of exogenous p300 would be significantly higher than
the
levels of transcription observed with only endogenous CBP/p300.
As expected, in the presence of additional p300, KID-ABP activated
transcription 20-fold over basal levels, confirming that the concentration of
endogenous
CBP/p300 in HEK293 cells is limiting (Figure 17C). Similarly, PPKID4P
activated
transcription 20-fold over basal levels in the presence of exogenous p300, a
2.7-fold
increase relative to PPKID4P-dependent transcription mediated by endogenous
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CBPlp300 alone. Likewise, PPKID6P activated transcription 15-fold over basal
levels
in the presence of additional p300, a 6-fold increase relative to PPKID6P-
dependent
transcription mediated by endogenous CBPlp300. Somewhat surprisingly, addition
of
p300 also increased transcription activation by PPKID6 in the absence of
S phosphorylation to levels 4.5-fold over basal transcription levels, whereas
PPKID6
failed to activate transcription in the presence of only endogeous CBP/p300.
Thus,
although unphosphorylated PPKID6 is perhaps best described as a weak
activation
domain, it nevertheless activates transcription via the CBP/p300 pathway.
These
results are consistent with a model in which PPKID4 and PPKID6, like CREB KID,
activate transcription by recruitment of CBP/p300 via the KID domain to the
promoters
where they are bound.
I. Transcription Inhibition by PPK.ID4P.
We have shown that transcriptional activation by PPKID4P and PPKID6P occur
via the same pathway as CREB KID, the CBP/p300 pathway. Therefore it would be
of
interest to show that these activatian domains indeed compete with each other
to
activate transcription in living cells. We compared the transcription
potential of
PPKID6P, PPKID4P and KID-ABP in the presence of increasing amounts of the
PPKID4P activation domain {without a DNA-binding domain). It is expected that
increasing amounts of the PPKID4P activation domain will bind the limited
supply of
CBP in the cells, thus preventing transcription via the CBPlp300 pathway.
Based on
the above results, we hypothesize that the PPKID4P activation domain will
inhibit
transcription of the phosphorylated PPKID and CREB ligands.
As expected, transcriptional activation by PPKID4P was significantly reduced
when a 2:1 ratio of PPKID4P activation domain:Gal4 DBD-PPKID4P was transfected
in
HEK~93 cells. Cotransfection of a 5-fold excess of PPKID4P activation domain
brought activation down to basal levels. Activation by KID-ABP was inhibited
when a
5:1 ratio of PPKID4P activation domain:Gal4 DBD-KID-ABP was transfected. With
a
10-fold excess of the PPKID4P activation domain, .KID-ABP activation was
completely
inhibited. Due to its low level of activation, PPKID6P activation is brought
down to
basal levels in the presence of only a 2-fold excess of the PPKID4P activation
domain.
In sum, the binding mode and orientation of two ligands for CBP KIX
(PPKID4P and PPKID6U) designed to mimic the natural ligand KID-ABP have been
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investigated. Binding affinity data with CBP KIX variants show that (despite
conformational differences) PPKID4P binds in the same hydrophobic pocket of
CBP
KIX as KID-ABP, possibly directed to this site by the phosphate group. Our
results
support a model in which PPKID4P folds into an aPP-like conformation upon
binding
S to CBP KIX. PPKID6U binds an overlapping yet distinct region of CBP KIX. The
distance separating the CBP KIX residues critical for binding this ligand
suggests that
PPKID6U binds in an open conformation. These ligands do not only bind their
target
with high aff nity in vitro, but they also function in mammalian cells.
PPKID4P and
PPKID6P function as transcriptional activators much like the transcription
factor CREB
after which they were modeled. These ligands can act as artificial activation
domains,
and have the potential to serve as tools in understanding the mechanism of
transcription
activation.
J. Experimental Section.
1) Expression and purification of CBP KIX and KIX mutants-- See above in
1S Example 19.
2) Peptide Synthesis and Modification-- PPKID4P, PPKID6U and KID-ABP
peptides were synthesized as described above in Example 19.
3) Fluorescence Polarization-- See above in Example 19.
4) Gal4 DBD-peptide constructs-- Peptides were cloned into a BamHI and SaII
digested pAli vector (a gift from John Frangioni) {Voss, et al., Anal Biochem
2002,
308, 364-372), which encodes the Gal4 DNA-binding domain and results in fusion
of
the peptide to the C-terminus to the Gal4 DBD. For PPKID4 inhibition
experiments,
PPKID4 was cloned into a BgIII and BamHI digested pAh vector, which removes
the
Gal4 DBD.
5) Cell Culture, Transfection and Luciferase Assays-- HEK293 cells (CRL-
1573 ATCC) were grown in DMEM containing 10°1° fetal bovine
serum. Cells were
plated in 24-well plates 24 hours prior to transfection. Cells were
transfected using
SuperFect transfection reagent (Qiagen) with 800 ng of total DNA. Included
were 5 ng
of peptide-Gal4 DNA-binding domain construct, 400 ng of SX Gal4 firefly
luciferase
reporter plasmid (a gift from John Frangioni), 20 ng of a promoterless Re~illa
luciferase plasmid (Promega) for normalization and pBluescript SK+ carnet DNA.
Where indicated forskolin was added to cell media 6 hours before harvested.
Cells
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WO 2005/044845 PCT/US2004/037210
were harvested and assayed 36 hours after transfection and Turner Designs
Model TD-
20/20 luminometer.
Example 21- Preparation of a universal miniature protein phase displa library
A combinatorial library designed to be used generally in the discovery and
engineering of miniature proteins can also be constructed using the methods of
the
invention. This universal library is designed to display a combinatorial set
of epitopes
to enable the recognition of nucleic acids, proteins or small molecules by a
miniature
protein without prior knowledge of the natural epitope used for recognition.
The
universal library optimally is formed by varying (at least about) six residues
on the
solvent-exposed face of aPP which do not contribute to the formation of the
hydrophobic aPP core. These residues of aPP include Tyr2l, Asn22, Asp22, G1n23
and
Asn26. All members of this universal library will retain the remarkable
stability and
compact structure of avian pancreatic polypeptide while introducing a diverse,
functional, solvent-exposed surface available for recognition. The number of
independent transformants (2.5 x 109 clones) required to cover sequence space
of a six-
membered library is experimentally feasible.
Incorporation By Reference
All publications and patents mentioned herein are hereby incorporated by
reference
in their entirety as if each individual publication or patent was specifically
and individually
indicated to be incorporated by reference.
While specific embodiments of the subject invention have been discussed, the
above specification is illustrative and not restrictive. Many variations of
the invention will
become apparent to those skilled in the art upon review of this specification
and the claims
below. The full scope of the invention should be determined by reference to
the claims,
along with their full scope of equivalents, and the specification, along with
such variations.
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