Note: Descriptions are shown in the official language in which they were submitted.
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STRUCTURE-BASED RATIONAL DESIGN OF COMPOUNDS TO INHIBIT
PAPILLOMAVIRUS INFECTION
FIELD OF THE INVENTION
The invention relates to methods of structure-based rational design of
compounds useful for inhibiting infection by papillomavirus.
BACKGROUND OF THE INVENTION
Papillomaviruses (PV) have been linked to widespread, serious human diseases,
especially carcinomas of the genital and oral mucosa. It is estimated that
there are
currently somewhere in the neighborhood of tens of millions of women who
suffer from
human papilloma virus (HPV) infection of the genital tract. Many of these
women
eventually develop cancer of the cervix. For example, it has been estimated
that about
twenty percent (20%) of all cancer related deaths in women worldwide are from
cancers
which are associated with HPV. It has also been estimated that 90% of all
cervical
cancer is linked to HPV.
Papillomaviruses induce benign, dysplastic and malignant hyperproliferations
of
skin or mucosal epithelium (see, for example, Mansur and Androphy, ( 1993)
Biochim
Biophys Acta 1155:323-345; Pfister (1984) Rev. Physiol. Biochem. Pharmacol.
99:111-
181; and Broker et al., ( I 986) Cancer Cells 4:17-36, for reviews of the
molecular,
cellular, and clinical aspects of the papillomaviruses). Almost 70 human
papillomavirus
types have been identified, and different papillomavirus types are known to
cause
distinct diseases, Poster, ( 1987) Adv. Cancer Res., 48 :113-147, Syrj anen, (
1984) Obstet.
Gvnecol. Survey 39:252-265. Human papillomaviruses (HPVs) are a heterogeneous
group of DNA tumor viruses associated with hypelplastic (warts, condylomata},
pre-
malignant and malignant lesions (carcinomas) of squamous epithelium. For
example,
HPV types 1 and 2 cause common warts, and types 6 and 11 cause warts of the
external
genitalia, anus and cervix. HPV, types 16, 18, 31 and 33 have been isolated
from the
majority of cervical cancers with HPV-16 present in about SO percent of all
cervical
cancers. These HPV's are referred to as "high risk". While HPV 6 and 11 are
the most
common isolates for cervical warts, these infections rarely progress to
invasive cancer,
and therefore these HPV's are referred to as "low risk".
Studies of viral gene expression in carcinomas suggest the importance of two
HPV encoded proteins, E6 and E7, in malignant development and these proteins
have
been shown to encode transforming and immortalizing activities. E6-induced
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tumorigenesis is thought to occur via multiple pathways (see Turek L., ( 1994)
Adv.
Virus Res. 44:305; Tommasino M. and Crawford L. ( 1995) BioEssays 17:509; Lee
J.M.
and Bernstein A. (1995) Cancer Metas. Rev. 14:149; Scheffner M. et al., (1993)
Cell
75:495; and Huibregste J. et al., (1993) Mol. Cell Biol. 13:775). In the p53-
dependent
pathway, the E6 protein associates with a human cellular factor, E6AP and in
the p53-
independent pathway, the E6 protein associates with a human cellular factor,
ERC55.
SUMMARY OF THE INVENTION
In general, the invention features, a method for evaluating a candidate
compound
for the ability to interact with, e.g., bind, an HPV E6 transforming protein.
The method
includes: supplying a three-dimensional structure for the E6 binding peptide
(E6bp);
supplying a three-dimensional structure for the candidate compound; and,
optionally,
comparing the three-dimensional structure of the candidate compound to the
three-
dimensional structure of the E6bp, thereby evaluating the candidate compound
for the
ability to interact with, e.g., bind the HPV E6 transforming protein.
In preferred embodiments, similarity in the structure of the candidate
compound
to the structure of the E6bp is indicative of the ability of the candidate
compound to
interact with the HPV E6 transforming protein.
In another aspect, the invention features, a method of providing or
identifying a
compound, preferably a compound which has the ability to interact with, e.g.,
bind, an
HPV E6 transforming protein. The method includes: supplying a three-
dimensional
structure for E6bp; supplying a three-dimensional structure for a candidate
compound;
optionally comparing the three-dimensional structure of the candidate compound
to the
three-dimensional structure of the E6bp; and optionally altering the structure
or altering
the spatial position of the structure of the candidate compound, thereby
providing or
identifying a compound, which preferably has the ability to interact with the
HPV E6
transforming protein.
In preferred embodiments, the altered structure of the candidate compound more
closely resembles the three-dimensional structure of E6bp, than does the
original
structure of the candidate compound.
In preferred embodiments, the method includes comparing the altered structure
of the candidate compound or the identified compound to the three-dimensional
structure of the E6bp. Preferably, the comparison can be performed by defining
an atom
equivalency in the candidate compound or the identified compound and the E6bp
three-
dimensional structures and comparing these atom equivalencies.
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In preferred embodiments, a second or further subsequent alteration is made in
the structure or the spatial position of the structure of the candidate
compound.
In preferred embodiments, the method includes defining an atom equivalency in
the candidate compound and the E6bp three-dimensional structures; and
performing a
fitting operation between the candidate compound and the E6bp three-
dimensional
structures.
In preferred embodiments, the method includes defining an atom equivalency in
the candidate compound and the E6bp three-dimensional structures; performing a
fitting
operation between the candidate compound and the E6bp three-dimensional
structures;
and analyzing the results of the fitting operation to compare the level of
similarity
between the candidate compound and the E6bp three-dimensional structures. For
example, the atom equivalencies can correspond to protein backbone atoms,
e.g., N, Ca,
C and O atoms. In preferred embodiments, the fitting operation can be a rigid
fitting
operation, e.g., the E6bp three-dimensional structure can be kept rigid and
the three-
I S dimensional structure of the candidate compound can be translated and
rotated to obtain
an optimum fit with the rigid target E6bp structure.
In preferred embodiments, the comparison between the candidate compound and
the E6bp three-dimensional structures can be performed computationally, e.g.,
by
calculating the root mean square deviation of a set of structural coordinates
in the
candidate compound from a set of structural coordinates in the E6bp, or
visually, e.g., by
visual inspection of the candidate compound and the E6bp three-dimensional
structures,
displayed in a graphical format.
In preferred embodiments, the candidate compound can have an a-helical
structure and the alteration can result in a change in the class of the a-
helix comprising
the structure of the candidate compound. For example, the a-helix comprising
the
structure of the candidate compound can be selected from the group consisting
of A, G,
and Y a-helices.
In preferred embodiments, the method includes creating a record of one or more
of the three-dimensional structures of the candidate compound, the altered
candidate
compound, the identified compound, and E6bp. The record can be encoded in the
form
of a machine-readable data storage medium. The three-dimensional structures
can be
displayed on a machine capable of displaying a graphical three-dimensional
representation.
In preferred embodiments, the method includes providing the identified
compound, e.g., chemically synthesizing the identified compound based on the
structure
identified using the methods described herein. In preferred embodiments, the
method
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includes assessing the biological activity of the identified compound. The
biological
activity of the identified compound can be assessed in vitro, e.g., in a GST-
E6 binding
assay or a two-hybrid assay, or in vivo, e.g., by applying the compound to a
cell line
(Hela, Caski, Siha) which expresses HPVE6 and examining the growth
characteristics of
the cells; or by its tumor suppression ability in an animal model for HPV
infection. In
preferred embodiments, the identified compound can be combined with a Garner
suitable
for introduction into an animal model, e.g., naturally derived or synthetic
polymers,
solvents, dispersion media, coatings, antibacterial and antifungal agents and
the like.
In preferred embodiments, the candidate compound can be altered so as to have
a
three-dimensional structure that is substantially similar to the three-
dimensional
structure of E6bp provided in figure 1, such that the candidate compound can
bind to the
HPV E6 transforming protein or portion thereof. For example, the candidate
compound
can be a peptide, a peptidomimetic, e.g., an isostere, an "inverso" or a
"retro-inverso"
peptide and the like, or a non peptide organic or inorganic compound.
In preferred embodiments, the identified compound associates with the HPV E6
transforming protein or a portion thereof, such that the ERC55 protein is
inhibited from
binding to E6. The association may be non-covalent or it may be covalent. The
association can be energetically favored by hydrogen bonding or van der Waals
or
electrostatic interactions.
In preferred embodiments, the three-dimensional structures can be supplied as
a
set of coordinates, defining the three-dimensional structures of the E6bp
molecule, the
candidate compound, the altered candidate compound and the identified compound
or as
a graphical three-dimensional representation of the E6bp molecule, the
candidate
compound, the altered candidate compound and the identified compound.
In another aspect, the invention features, a machine-readable data storage
medium, including a data storage material encoded with a set of NMR derived
coordinates which define the three-dimensional structure of the E6bp molecule.
The
storage medium can be used in methods of the invention.
In yet another aspect, the invention features, a machine-readable data storage
medium, including a data storage material encoded with machine readable data
which,
when used with a machine programmed with instructions for using the data, is
capable
of displaying a graphical three-dimensional representation of the E6bp
molecule. The
storage medium can be used in methods of the invention.
In another aspect, the invention features, a method of treating a subject at
risk for
infection by a HPV. For example, a subject at risk for an HPV induced cancer,
e.g.,
cervical cancer, can be treated. The method includes: administering to a subj
ect a
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therapeutically effective amount of a compound other than an E6bp fragment,
wherein
the compound has a structure sufficiently duplicative of that of Figure 1, so
as to bind to
the HPV E6 transforming protein and prevent its interaction with the ERC55
protein,
thereby treating a subject at risk for infection by a HPV.
In preferred embodiments, 50, 60, 70 and more preferably 80, 90 or 100% of the
HPV E6 protein, present in the cell, can be bound to the compound and can,
therefore,
be unable to bind to ERC55 and induce cellular transformation.
In another aspect, the invention features, a compound, other than an E6bp or
ERC55 fragment, having a three-dimensional structure substantially similar to
the three-
dimensional structure of E6bp provided in Figure 1, such that the candidate
compound
can bind to the HPV E6 transforming protein.
In preferred embodiments, the candidate compound has a structure sufficiently
duplicative of the three-dimensional structure E6bp provided in Figure l, such
that the
candidate compound can bind to the HPV E6 transforming protein, with an
affinity
which is at least half that of E6bp. The dissociation constant (Kd) for the E6-
compound
complex, is less than 100, 50 or 10 times the Kd of the E6-E6bp complex, and
more
preferably less than the Kd of the E6-E6bp complex.
In preferred embodiments, the candidate compound is more stable (e.g., more
resistant to proteolytic degradation) than E6bp or ERC55.
In another aspect, the invention features, a composition comprising a
compound,
other than an E6bp or ERC55 fragment, having a three-dimensional structure
sufficiently duplicative of the three-dimensional structure of E6bp provided
in Figure 1,
such that the compound can bind to the HPV E6 transforming protein, and a
carrier
macromolecule suitable for the administration of the composition to a subject.
In another aspect, the invention features, a method of providing or
identifying a
compound, preferably a compound which can interact with, e.g., bind E6bp. The
method includes: supplying a three-dimensional structure for the E6bp
molecule;
supplying a three-dimensional structure for the candidate compound; optionally
comparing the three-dimensional structure of the candidate compound to the
three-
dimensional structure of the E6bp; optionally altering the structure or
altering the spatial
position of the structure of the candidate compound; optionally comparing the
altered
structure of the candidate compound to the structure of the E6bp, thereby
providing or
identifying a compound, preferably a compound which can interact with, e.g.,
bind
E6bp.
3 5 In preferred embodiments, comparing includes performing a fitting
operation.
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In another aspect, the invention features, a method of evaluating the ability
of a
candidate compound to interact with, e.g., bind an E6bp molecule. The method
includes: supplying a three-dimensional structure for the E6bp molecule;
supplying a
three-dimensional structure for the candidate compound; and performing a
fitting
operation between the three-dimensional structures of the candidate compound
and the
E6bp molecule, thereby evaluating the ability of the candidate compound to
interact
with the E6bp molecule.
In preferred embodiments, the method includes analyzing the results of the
fitting operation to quantify the association between the candidate compound
and the
E6bp molecule.
In preferred embodiments, the method includes defining an atom equivalency in
the candidate compound and the E6bp molecule three-dimensional structures. For
example, the atom equivalencies can correspond to protein backbone atoms,
e.g., N, Ca,
C and O atoms. In preferred embodiments, the fitting operation can be a rigid
fitting
operation, e.g., the E6bp three-dimensional structure can be kept rigid and
the three-
dimensional structure of the candidate compound can be translated and rotated
to obtain
an optimum fit with the rigid target E6bp structure.
In preferred embodiments, the fitting operation can be performed
computationally, e.g., by calculating the root mean square deviation of a set
of structural
coordinates in the candidate compound from a set of structural coordinates in
the E6bp,
or visually, e.g., by visual inspection of the candidate compound and the E6bp
three-
dimensional structures, displayed in a graphical format.
In preferred embodiments, the method includes altering the structure or
altering
the spatial position of the structure of the candidate compound.
In preferred embodiments, the method includes creating a record of one or more
of the three-dimensional structures of the candidate compound, the altered
candidate
compound, the identified compound and the E6bp molecule. The record can be
encoded
in the form of a machine-readable data storage medium. The three-dimensional
structures can be displayed on a machine capable of displaying a graphical
three-
dimensional representation.
In preferred embodiments, the method includes providing the identified
compound, e.g., chemically synthesizing the identified compound based on the
structure
identified using the methods described herein. In preferred embodiments, the
method
includes assessing the biological activity of the identified compound. The
biological
activity of the identified compound can be assessed in vitro, e.g., in a GST-
E6bp or a
GST-ERCSS binding assay or a two-hybrid assay, or in vivo, e.g., by its tumor
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suppression ability in an animal model for HPV infection. In preferred
embodiments,
the identified compound can be combined with a carrier suitable for
introduction into an
animal model, e.g., naturally derived or synthetic polymers, solvents,
dispersion media,
coatings, antibacterial and antifungal agents and the like.
In preferred embodiments, the candidate compound can have an a-helical
structure and the alteration can result in a change in the class of the a-
helix comprising
the structure of the candidate compound. For example, the a-helix comprising
the
structure of the candidate compound can be selected from the group consisting
of A, G,
and Y a-helices.
In preferred embodiments, the altered compound can associate with the E6bp
molecule with a higher affinity.
In preferred embodiments, evaluating includes determining the ability of a
compound to interact with, e.g., bind the E6bp molecule. Evaluation can be
performed
computationally, e.g., by calculating the root mean square deviation of a set
of structural
coordinates in the candidate compound from a set of structural coordinates in
the E6bp,
or visually, e.g., by visual inspection of the candidate compound and the E6bp
three-
dimensional structures, displayed in a graphical format.
In preferred embodiments, the candidate compound includes a compound which
can be altered so as to have a three-dimensional structure that is suitable
for associating
with E6bp and, therefore, with ERC55. For example, the candidate compound can
be a
peptide, a peptidomimetic, e.g., an isostere, an "inverso" or a "retro-
inverso" peptide and
the like, or a non peptide organic compound.
In preferred embodiments, the candidate compound associates with the E6bp,
such that the ERCS S protein is inhibited from binding to E6. The association
may be
non-covalent or it may be covalent. The association can be energetically
favored by
hydrogen bonding or van der Waals or electrostatic interactions.
In preferred embodiments, the three-dimensional structures can be supplied as
a
set of coordinates, defining the three-dimensional structures of the E6bp
molecule, the
candidate compound, the altered candidate compound and the identified compound
or as
a graphical three-dimensional representation of the E6bp molecule, the
candidate
compound, the altered candidate compound and the identified compound.
In another aspect, the invention features, a method of treating a subject at
risk for
infection by a HPV. For example, a subject at risk for an HPV induced cancer,
e.g.,
cervical cancer, can be treated. The method includes: administering to a subj
ect a
therapeutically effective amount of a compound, wherein the compound
associates with
E6bp with an affinity so as to prevent the interaction between the ERC55 and
the HPV
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E6 protein, thereby treating a subject at risk for infection by a HPV.
Preferred
compounds are provided by the methods described herein.
In another aspect, the invention features, a method of modeling the region of
the
HPV E6 protein which binds ERC55. The method includes: supplying a three-
dimensional structure for an E6bp molecule and supplying a structure, which is
complementary to the structure of the E6bp molecule, thereby modeling the
region of
the HPV E6 protein which binds ERC55.
The molecular modeling techniques, described herein can be used to construct a
structure, which is complementary to the E6bp three-dimensional structure. By
"complementary", is meant a structure, which is complementary to one or more
of: (a)
the shape, (b) the electrostatic properties or (c) the hydrophobicity of the
E6bp three-
dimensional structure. While not wishing to be bound by theory, E6bp may be
complementary in shape to a critical portion of E6. Thus, something
complementary to
E6bp mimics the structure of E6. The complementary structure need not be
translated
into a real molecule, but can be used in the computational or computer based
methods
described herein, to identify a compound which has the ability to interact
with the HPV
E6 transforming protein.
In preferred embodiments, the method includes creating a record of the three-
dimensional structure of the E6bp molecule and its complementary structure.
The
record of the three-dimensional structure of the E6bp molecule and its
complementary
structure can be encoded in the form of a machine-readable data storage
medium. The
three-dimensional structures can be displayed on a machine capable of
displaying a
graphical three-dimensional representation of a structure.
In preferred embodiments, the three-dimensional structure can be supplied as a
set of coordinates, defining the three-dimensional structure of the E6bp
molecule and its
complementary structure or as a graphical three-dimensional representation of
the E6bp
molecule and its complementary structure.
As used herein, the term "comparing" refers to examining a quality, e.g.,
three-
dimensional structure, hydrophobicity, steric bulk, electrostatic properties,
bond angles,
size or molecular composition of a compound, in order to identify resemblances
or
differences between two structures.
As used herein, the term "altering the structure" refers to altering the
intrinsic
properties, e.g., three-dimensional structure, hydrophobicity, steric bulk,
electrostatic
properties, bond angles, size or molecular composition of a compound. In a
peptide
molecule, the alteration can include an amino acid substitution or the
introduction of a
non-peptide molecule or bond in the structure of the candidate compound. The
non-
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peptide molecule or bond can comprise a peptidomimetic entity, e.g., a
peptidomimetic
molecule or bond.
As used herein, the term "altering the spatial position" refers to changing
the
orientation of or translating the structure of the candidate compound,
relative to a pre-
y defined reference, e.g., relative to the structure of the E6bp molecule. For
example, the
structure of the candidate compound can be rotated, e.g., 30, 60, 90, 120 or
180° relative
to the structure of the E6bp molecule.
As used herein, the term "atom equivalencies" refers to a set of conserved
residues between two structures, defined such that they allow direct
comparison of the
structures being compared. For example, the atom equivalencies can correspond
to
protein backbone atoms, e.g., N, Ca, C and O atoms.
As used herein, the term "fitting operation" refers to the process by which, a
working structure (i.e. a compound) is translated and rotated to obtain an
optimum fit
with the target E6bp structure. The fitting operation can use a least squares
fitting
algorithm that computes the optimum translation and rotation to be applied to
the
moving compound structure, such that the root mean square difference of the
fit over the
specified pairs of equivalent atoms is an absolute minimum. This number, given
in
angstroms, can be reported by a computer software.
As used herein, the term "root mean square deviation" refers to the square
root of
the arithmetic mean of the squares of the deviations from the mean. It is a
way to
express the deviation or variation from a trend or object. For purposes of
this invention,
the "root mean square deviation" defines the variation in a set of atom
equivalencies of a
compound from a set of atom equivalencies of the E6bp molecule, as defined by
the
structure coordinates of the E6bp molecule described herein.
As used herein, the term "least squares" refers to a method based on the
principle
that the best estimate of a value is that in which the sum of the squares of
the deviations
of observed values is a minimum.
The methods of the invention allow rapid and efficient design and evaluation
of
compounds useful for inhibiting infection by papillomavirus.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
DETAILED DESCRIPTION
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings are first briefly described.
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Figure 1 is a depiction of the three-dimensional structure of E6bp. The
protein
backbone is shown as a blue ribbon. Hydrophobic amino acid side chains are
shown in
yellow, and polar side-chains in blue. Calcium ions are shown as spheres.
Figure 2 is an amide-amide region of the 2D NOESY illustrating the structure
of
the E6bp molecule. Intense amide to amide proton contacts, indicative of a
helices, are
indicated by residue number. Residues 13 to 16, shown above the diagonal, form
an N
terminal helix, whereas residues 22 to 27, shown below the diagonal, for a C-
terminal
helix.
Figure 3 is an illustration of ligand design. In this example, the candidate
compound phenylarginine was built onto two exposed amino acids residues
(glutamate
16 and leucine 19) on the C-terminal a helix of the E6bp protein. The
hydrocarbons of
the ligand are indicated by G, whereas those of the protein are indicated by
Y. Oxygen
atoms are indicated by R, nitrogens by B, and polar hydrogens by W. The
compound
was designated with the LUDI feature feature of the molecular modeling program
INSIGHTII, and the fit to the protein was optimized using the DOCK module.
This
figure illustrates the method for design of novel inhibitors to
papillomavirus, described
herein.
Figure 4 is a depiction of various sequences illustrating that the E6 binding
domains is a short a-helical peptide and that the E6 binding region of E6BP is
found in
other E6-binding proteins. Further illustrated in this Figure are the results
from an
analysis of the structure of E6bp, based on site-directed mutagenesis.
E6 Signal Transduction Pathway
E6-induced tumorigenesis occurs via two pathways. In the p53-dependent
pathway, the E6 protein associates with a human cellular factor, E6AP. The E6-
E6AP
complex directs p53 for rapid degradation via the ubiquitin-mediated
proteolytic
pathway (Lee J.M. and Bernstein A. (1995) Cancer Metas. Rev. 14:149; Scheffner
M. et
al., (1993) Cell 75:495; and Huibregste J. et al., (1993) Mol. Cell Biol.
13:775). Loss of
p53 protein correlates with the loss in its tumor suppressor functions. An 18
amino acid
residue peptide fragment, E6ap, is the minimal region of E6AP that binds E6
(Huibregste J. et al., (1993) Mol. Cell Biol. 13:4918).
E6-induced tumorigenesis is also p53-independent (Storey A. et al., (1995)
Oncogene 11:653), and a different target protein, ERC55, has been demonstrated
to bind
E6 (Chen J.J. et al., (1995) Science 269:529). ERC55 and p53 compete for
binding to
E6, consistent with alternate roles in tumorigenesis. The mechanism of ERC55
function
appears to involve alteration of keratinocyte differentiation (Sherman L. and
Schlegel R.
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( 1996) J. Virol. 70:3269; and Reiss M. et al., ( 1989) Cancer Commun. 1:75;
Chen J.J. et
al., ( 1995 ) Science 269:529; and Howley P.M. ( 1996} Field Virol. Chapter
65, 2nd
edition). A 25 amino acid segment of ERC55, called E6bp, has been found to be
necessary and sufficient for binding to E6.
The sequence of E6ap is homologous to E6bp, as shown in the following Table.
TABLE I
E6ap IPESSELTLQELLGEERR (SEQ LD. NO:1 )
**~*~*~~*
E6bp ALEEHDKNGDGFVSLEEFLGDYRWD (SEQ LD. N0:2)
The * symbol indicates homologous amino acids, whereas the ~ symbol denotes
identical amino acids.
Structural
The inventors have solved the three-dimensional structure of an E6bp molecule,
using one-and two-dimensional NMR Spectroscopy. Importantly, this has
provided, for
the first time, information about the three-dimensional structure of the E6bp
molecule.
This information is of significant utility in fields such as drug discovery.
An
understanding of the structure of the E6bp molecule, a 25 amino acid region in
the
ERC55 protein sufficient for binding to the HPV E6 transforming protein,
allows the
design of drugs which interact with the HPV E6 transforming protein. As a
result, this
information is useful for designing inhibitors of the E6-ERC55 interaction and
therefore, drugs for fighting papillomavirus infection.
Candidate Compounds
Candidate compounds can be agents which can be altered so as to have a three-
dimensional structure that is substantially similar to the three-dimensional
structure of
E6bp, provided in Figure 1, such that the agent can bind to the HPV E6
transforming
protein or portion thereof. Preferably, the altered candidate compound can
bind to E6
with an affinity which is at least 10, 50, 100, 150, 200 or 500% as strong as
the affinity
with which E6bp binds to E6. Candidate compounds can also be agents which can
be
altered so as to have a three-dimensional structure that is suitable for
associating with
the E6bp molecule.
For example, the candidate compound can be a peptide or a peptidomimetic.
Examples of peptidomimetics include peptidic compounds in which the peptide
backbone is substituted with one or more benzodiazepine molecules (see e.g.,
James,
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G.L. et al., 1993, Science 260:1937-1942, the contents of which are
incorporated herein
by reference), peptides in which all L-amino acids are substituted with the
corresponding
D-amino acids, and "retro-inverso" peptides (see U.S. Patent No. 4,522,752 by
Sisto, the
contents of which are incorporated herein by reference).
The term mimetic, and in particular, peptidomimetic, includes isosteres. The
term "isostere" as used herein, includes a chemical structure that can be
substituted for a
second chemical structure because the steric conformation of the first
structure fits a
binding site specific for the second structure. The term specifically includes
peptide
back-bone modifications (e.g., amide bond mimetics). Such modifications
include
modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete
replacement of the amide bond, extensions, deletions or backbone cross links.
Several
peptide backbone modifications are known, including y~[CH2S], y~[CH,NH], yr
[CSNH2], ~[NHCO], to[COCH2], and yr[(E) or (Z) CH=CH]. In the nomenclature
used
above, y indicates the absence of an amide bond. The structure that replaces
the amide
group is specified within the brackets.
Other possible modifications include an N-alkyl (or aryl) substitution (y~
(CONK]), backbone cross linking to construct lactams and other cyclic
structures,
substitution of all D-amino acids for all L-amino acids within the candidate
compound
("inverso" compounds) or retro-inverso amino acid incorporation (~r[NHCO]). By
"inverso" is meant replacing L-amino acids of a sequence with D-amino acids,
and by
"retro-inverso" or "enantio-retro" is meant reversing the sequence of the
amino acids
("retro") and replacing the L-amino acids with D-amino acids. For example, if
the
parent peptide is Thr-Ala-Tyr, the retro modified form is Tyr-Ala-Thr, the
inverso form
is thr-ala-tyr, and the retro-inverso form is tyr-ala-thr (lower case letters
refer to D-
amino acids). Compared to the parent peptide, a retro-inverso peptide has a
reversed
backbone while retaining substantially the original spatial conformation of
the side
chains, resulting in a retro-inverso isomer with a topology that closely
resembles the
parent peptide. (See Goodman et al. "Perspectives in Peptide Chemistry" pp.
283-294,
1981, and U.S. Patent No. 4,522,752, the contents of which are incorporated
herein by
reference).
The candidate compound can also be a non peptide organic compound prepared
as described in WO 9504277, the contents of which are incorporated herein by
reference, as well as a steroid, a carbohydrate, a lipid and the like.
The candidate compound can be selected from a database of three-dimensional
structures of known compounds. The three-dimensional structures in the
database can
be either experimentally determined, e.g., crystal structures from the
Cambridge
CA 02273186 1999-06-02
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structural database (see Allen et al., J. Chem. Inf. Comput. Sci. 31: 187-204,
1991, the
contents of which are incorporated herein by reference) or computationally
generated,
e.g., using rule-based programs such as CONCORD (see Pearlman, R. S., Chem.
Des.
Auto. News, 2:1-7, 1987, the contents of which are incorporated herein by
reference).
The candidate compounds can also be designed de novo; e.g., by piecing
together
or assembling molecular fragments to create compounds which: (a) have a three-
dimensional structure that is substantially similar to the three-dimensional
structure of
E6bp provided in figure l, such that the created compound can bind to the HPV
E6
transforming protein or portion thereof or (b) have a three-dimensional
structure that is
suitable for associating with the E6bp molecule. For example, the GROW
algorithm
(Moon, J.B., et al., Proteins: Struct. Funct. Genet 11:314-328,1991, the
contents of
which are incorporated herein by reference), or the LUDI program (Bohm, H.-J.
J
Comput Aided Mol Design 6:61-78,1992, the contents of which are incorporated
herein
by reference) can be used.
1 S Other useful programs to aid one of skill in the art in assembling
molecular
components to create compounds include: (1) CAVEAT, described in Bartlett et
al., In
"Molecular Recognition in Chemical and Biological Problems", Special Pub.,
Royal
Chem. Soc., 78, 182-196,1989, the contents of which are incorporated herein by
reference; (2) 3D Database systems such as MACCS-3D (MDL Information Systems,
San Leandro, CA). The use of these systems is described in Martin et al., J.
Med.
Chem., 35, 2145-2154,1992, the contents of which are incorporated herein by
reference;
and (3) HOOK {available from Molecular Simulations, Burlington, MA).
Other molecular modeling techniques may also be employed in accordance with
this invention. See e.g., Cohen et al., J. Med. Chem., 33, 883-894,1990, Naiva
et al.,
Current Opinions in Structural Biology, 2, 202-210,1992, the contents of which
are
incorporated herein by reference.
Once a compound has been designed by the above methods, its similarity to the
three-dimensional structure of the E6bp peptide may be evaluated.
Machine Read;~le Storage Medium
In order to use the NMR derived structure coordinates for the E6bp peptide, it
is
preferable to convert them into a three-dimensional representation. This can
be achieved
through the use of commercially available software which is capable of
generating three-
dimensional graphical representations of molecules or portions thereof from as
set of
structure coordinates.
Evaluation and Design of Candidate Comy~,ounds
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The invention allows the use of molecular design techniques to design and
evaluate candidate compounds, including inhibitory compounds, e.g., candidate
compounds having a three-dimensional structure that is: (a) substantially
similar to the
three-dimensional structure of E6bp provided in figure 1, such that the
candidate
compounds can bind to the HPV E6 transforming protein or portion thereof or
(b)
suitable for associating with the E6bp molecule.
A potential compound which can bind to the HPV E6 transforming protein or
portion thereof can be evaluated by means of a series of steps in which
compounds are
screened and selected for their similarity to the three-dimensional structure
of the E6bp
molecule.
One skilled in the art can use one of several methods to screen compounds for
their similarity to the three-dimensional structure of the E6bp molecule. This
process
may begin by visual inspection of, for example, the three-dimensional
structure of the
candidate compound in comparison to the three-dimensional structure of the
E6bp
molecule on a computer screen, wherein the three-dimensional structure of the
E6bp
molecule is generated from the machine-readable storage medium.
Various computational analyses can also be used to determine whether a
compound is sufficiently similar to the three-dimensional structure of the
E6bp
molecule. Such analyses can be carried out in current software applications,
such as the
Molecular Similarity application of QUANTA (Molecular Simulations Inc.,
Waltham,
MA) version 3.3, and as described in the accompanying User's Guide, Volume 3
pg.
134-135, the contents of which are incorporated herein by reference.
The Molecular Similarity application permits comparisons between different
structures, different conformations of the same structure, and different parts
of the same
structure. The procedure used in Molecular Similarity to compare structure is
divided
into four steps: 1 ) loading the structures to be compared; 2) defining the
atom
equivalences in these structures; 3) performing a fitting operation; and 4)
analyzing the
results. -
Each structure can be identified by a name. The E6bp structure can be
identified
as the target (i.e., the fixed structure); the candidate compound structures
can be working
structures (i.e., moving structures). Since atom equivalency within QUANTA is
defined
by user input, equivalent atoms can be defined as protein backbone atoms (N,
Ca,, C and
O) for all conserved residues between the two structures being compared. The
process
can be aided by color-coding the different parts of the molecules.
Rigid fitting operations can be used. When a rigid fitting method is used, the
working compound structure is translated and rotated to obtain an optimum fit
with the
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target E6bp structure. The fitting operation uses a least squares fitting
algorithm that
computes the optimum translation and rotation to be applied to the moving
compound
structure, such that the root mean square difference of the fit over the
specified pairs of
equivalent atom is an absolute minimum. This number, given in angstroms, can
be
reported by QUANTA.
Preferred candidate structures are those having a set of structure coordinates
with
a root mean square deviation of conserved residue backbone atoms (e.g., N, Ca,
C, O) of
less than 1.5 A when superimposed, using backbone atoms, on the relevant
structure
coordinates listed in Figure 2 are considered identical. More preferably, the
root mean
square deviation is less than 1.0 ~, and even more preferably, the root mean
square
deviation is less than 0.5 ~.
Candidate compounds can also be evaluated for their ability to associate with
the
E6bp molecule. One skilled in the art may use one of several methods to screen
compounds for their ability to associate with the E6bp molecule. This process
may
begin by visual inspection of, for example, the E6bp molecule on a computer
screen
based on the NMR derived data shown in Figure 2, and generated from the
machine-
readable storage medium. Selected compounds may then be positioned in a
variety of
orientations, or docked, within the E6bp molecule. Docking may be accomplished
using
software such as Quanta and Sybyl, followed by energy minimization and
molecular
dynamics with standard molecular mechanics force fields, such as CHARMM and
AMBER. Specialized computer programs may also assist in the process of
selecting
fragments or chemical entities. These include: 1. AUTODOCK (D.S. Goodsell et
al.,
"Automated Docking of Substrates to Proteins by Simulated Annealing" Proteins:
Structure. Function, and Genetics, 8, pp. 195-202 (1990)). DOCK is available
from
University of California, San Francisco, CA. 2. DOCK, (LD. Kuntz et al., J.
Mod, Biol.,
161, pp. 269-288 (1982), the contents of which are incorporated herein by
reference).
DOCK is available from University of California, San Francisco, CA.
~y~thesis of Identified Com on unds
Once a compound has been evaluated by the above methods, it can be prepared
by standard techniques known in the art. Peptides can be synthesized using
standard
techniques such as those described in Bodansky, M. P~incinles of P tide
Synthesis,
Springer Verlag, Berlin ( 1993) and Grant, G.A (ed.). ~vnthetic Pe~ides: A
User's
Guide, W.H. Freeman and Company, New York (1992). Automated peptide
synthesizers are commercially available (e.g., Advanced ChemTech Model 396;
Milligen/ Biosearch 9600). Approaches to designing peptide analogs are also
known in
the art. For example, see Farmer, P.S. in Drug Desig~0 (E.J. Ariens, ed.)
Academic
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Press, New York, 1980, vol. 10, pp. 119-143; Ball. J.B. and Alewood, P.F.
(1990) J.
Mol. Recognition 3_:55; Morgan, B.A. and Gainor, J.A. (1989) Ann. Rep. Med.
Chem.
24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270, the
contents of all
of which are incorporated herein by reference.
Alternatively, peptide compounds can be prepared according to standard
recombinant DNA techniques using a nucleic acid molecule encoding the peptide.
A
nucleotide sequence encoding the peptide can be determined using the genetic
code and
an oligonucleotide molecule having this nucleotide sequence can be synthesized
by
standard DNA synthesis methods (e.g., using an automated DNA synthesizer).
Alternatively, a DNA molecule encoding a peptide compound can be derived from
the
corresponding natural gene or cDNA (e.g., using the polymerase chain reaction
andlor
restriction enzyme digestion) according to standard molecular biology
techniques.
To facilitate expression of a peptide compound in a host cell by standard
recombinant DNA techniques, the isolated nucleic acid encoding the peptide is
1 S incorporated into a recombinant expression vector. As used herein, the
term "vector"
refers to a nucleic acid molecule capable of transporting another nucleic acid
to which
it has been linked. One type of vector is a "plasmid", which refers to a
circular
double stranded DNA loop into which additional DNA segments may be ligated.
Another type of vector is a viral vector, wherein additional DNA segments may
be
ligated into the viral genome. Certain vectors are capable of autonomous
replication
in a host cell into which they are introduced (e.g., bacterial vectors having
a bacterial
origin of replication and episomal mammalian vectors). Other vectors (e.g.,
non-
episomal mammalian vectors) are integrated into the genome of a host cell upon
introduction into the host cell, and thereby are replicated along with the
host genome.
Moreover, certain vectors are capable of directing the expression of genes to
which
they are operatively linked. Such vectors are referred to herein as
"recombinant
expression vectors" or simply "expression vectors". In general, expression
vectors of
utility in recombinant DNA techniques are often in the form of plasmids. In
the
present specification, "plasmid" and "vector" may be used interchangeably as
the
plasmid is the most commonly used form of vector. However, such other forms of
expression vectors, such as viral vectors, which serve equivalent functions
may also
be used to express a peptide compound.
The nucleotide sequence encoding the peptide compound can be operatively
linked to one or more regulatory sequences, selected on the basis of the host
cells to be
used for expression. The term "operably linked" is intended to mean that the
sequences
encoding the peptide compound are linked to the regulatory sequences) in a
manner that
*rB
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allows for expression of the peptide compound. The term "regulatory sequence"
is
intended to include promoters, enhancers and other expression control elements
(e.g.,
polyadenylation signals). Such regulatory sequences are described, for
example, in
Goeddel; Gene Expression Technology: Methods in Enzymology ~, Academic Press,
San Diego, CA (1990), the content of which are incorporated herein by
reference.
Regulatory sequences include those that direct constitutive expression of a
nucleotide
sequence in many types of host cells, those that direct expression of the
nucleotide
sequence only in certain host cells (e.g., tissue-specific regulatory
sequences) and those
that direct expression in a regulatable manner (e.g., only in the presence of
an inducing
agent). It will be appreciated by those skilled in the art that the design of
the expression
vector may depend on such factors as the choice of the host cell to be
transformed, the
level of expression of peptide compound desired, and the Like. The peptide
compound
expression vectors can be introduced into host cells to thereby produce
peptide
compounds encoded by nucleic acids.
The recombinant expression vectors can be designed for expression of peptide
compounds in prokaryotic or eukaryotic cells. For example, peptide compounds
can be
expressed in bacterial cells such as E. coli, insect cells (using baculovirus
expression
vectors) yeast cells or mammalian cells. Suitable host cells are discussed
further in
Goeddel, Gene Expression Technology: Methods in Enzymology 1,,~$~5, Academic
Press,
San Diego, CA ( 1990). Alternatively, the recombinant expression vector may be
transcribed and translated in vitro, for example using T7 promoter regulatory
sequences
and T7 polymerise. Examples of vectors for expression in yeast S. cerevisiae
include
pYepSecl (Baldari et al., (1987) EMBO J. 6_:229-234), pMFa (Kurjan and
Herskowitz,
(1982) Cell x_0:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), and
pYES2
(Invitrogen Corporation, San Diego, CA). Baculovirus vectors available for
expression
of peptide compounds in cultured insect cells (e.g., Sf 9 cells) include the
pAc series
(Smith et al., (1983) Mol. Cell. Biol. 3_:2156-2165) and the pVL series
(Lucklow, V.A.,
and Summers, M.D., (1989) Virology ~( :31-39).
Examples of mammalian expression vectors include pCDM8 (Seed, B., ( 1987)
Nature .2:840) and pMT2PC (Kaufman et al. (1987), EMBO J. x:187-195). When
used in mammalian cells, the expression vector's control functions are often
provided
by viral regulatory elements. For example, commonly used promoters are derived
from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
In addition to the regulatory control sequences discussed above, the
recombinant expression vector may contain additional nucleotide sequences. For
example, the recombinant expression vector may encode a selectable marker gene
to
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identify host cells that have incorporated the vector. Such selectable marker
genes
are well known in the art. Moreover, to facilitate secretion of the peptide
compound
from a host cell, in particular mammalian host cells, the recombinant
expression
vector preferably encodes a signal sequence operatively linked to sequences
encoding
the amino-terminus of the peptide compound such that upon expression, the
peptide
compound is synthesized with the signal sequence fused to its amino terminus.
This
signal sequence directs the peptide compound into the secretory pathway of the
cell
and is then cleaved, allowing for release of the mature peptide compound
(i.e., the
peptide compound without the signal sequence) from the host cell. Use of a
signal
sequence to facilitate secretion of proteins or peptides from mammalian host
cells is
well known in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell,
including
calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated
transfection, lipofection, electroporation, microinjection and viral-mediated
transfection.
Suitable methods for transforming or transfecting host cells can be found in
Sambrook
et al. (Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring
Harbor
Laboratory press ( 1989)), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending upon
the
expression vector and transfection technique used, only a small fraction of
cells may
integrate the foreign DNA into their genome. In order to identify and select
these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene encoding the
peptide
compound. Preferred selectable markers include those that confer resistance to
drugs,
such as 6418, hygromycin and methotrexate. Nucleic acid encoding a selectable
marker
may be introduced into a host cell on the same vector as that encoding the
peptide
compound or may be introduced on a separate vector. Cells stably transfected
with the
introduced nucleic acid can be identified by drug selection {e.g., cells that
have
incorporated the selectable marker gene will survive, while the other cells
die).
Assessment of the Biological Activity of Identified Com~o3~nds
Once a compound has been identified and synthesized, its biological activity
can
be assessed. For example, an assay can be used to assess the ability of a
compound to
inhibit binding between ERC55 (i.e., E6bp) and the HPV E6 transforming
protein. A
variety of assays are available and readily apparent to the skilled artisan.
For example,
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in one such biological evaluation assay the identified compound can be
contacted with
an isolated and purified ERC55 protein. The mixture of the candidate compound
and
ERC55 can then be added to a composition containing the E6 protein but which
does not
contain ERC55. Detection and quantification of labelled E6/ERC55 complexes
provides
a means for determining the candidate compound's efficacy at inhibiting
complex
formation between the papillomavirus E6 protein and the ERC55 protein. A
control
assay can also be performed to provide a baseline for comparison. In the
control assay,
isolated and purified ERC55 is added to a composition containing the E6
protein, and
the formation of an E6/ERC55 complex is quantitated in the absence of the test
compound.
Complex formation between ERC55 and E6 may be detected by a variety of
other methods as well. For example, glutathione-S-transferase/E6 (GST/E6)
fusion
proteins can be adsorbed onto glutathione sepharose beads which can then be
combined
with an 35S-labeled ERC55 protein and incubated under conditions conducive to
complex formation, [e.g., at 4°C in a buffer of 25 mM Tris-HCl (pH
7.2), 50 mM NaCI
and 0.2% NP-40]. Following incubation, the beads can be washed to remove any
unbound ERC55, and the sepharose bead-bound radiolabel can be determined
directly
(e.g. beads placed in scintilant), or in the superntantant after the E6/ERC55
complexes
are dissociated (e.g. by treatment with DTT). The supernatant containing the
complexes
can be separated by SDS-PAGE gel before detection.
Additionally, ERC55 or E6bp can be used to generate a two-hybrid assay, as
described in U.S. Patent No: 5,283,317; Zervos et al. (1993) Cell 72:223-232;
Madura et
al. ( / 993) J Biol Chem 268:12046-12054; Bartel et al. ( 1993) Biotechniques
14:920-
924; and Iwabuchi et al. (1993) Oncogene 8:1693-1696), for subsequently
assessing the
ability of compounds to disrupt binding of ERC55 or E6bp to E6. The
interaction trap
assay relies on reconstituting in vivo a factional transcriptional activator
protein from
two separate fusion proteins, one of which comprises the DNA-binding domain of
a
transcriptional activator fused to an E6 protein. The second fusion protein
comprises a
transcriptional activation domain (e.g. able to initiate RNA polymerase
transcription)
fused to ERC55 or E6bp. When the E6 and ERC55 or E6bp proteins interact, the
two
domains of the transcriptional activator protein are brought into sufficient
proximity as
to cause transcription of a reporter gene. For example, Saccharomyces
cerevisiae YPB2
cells can be transformed simultaneously with a plasmid encoding a GAL4db-E6
fusion
and with a plasmid encoding the GAL4ad domain fused to ERC55 or E6bp.
Moreover,
the strain is transformed such that the GAL4-responsive promoter drives
expression of a
phenotypic marker. For example, the ability to grow in the absence of
histidine depends
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on the expression of the HIS3 gene. When the HIS3 gene is placed under the
control of
a GAL4-responsive promoter, relief of this auxotrophic phenotype indicates
that a
functional GAL4 activator has been reconstituted through the interaction of E6
and
ERC55 or E6bp. Thus, a compound able to inhibit an ERC55 or E6bp interaction
with
E6 will result in yeast cells unable to grow in the absence of histidine.
Alternatively, the
phenotypic marker (e.g. instead of the HIS3 gene) can be one which provides a
negative
selection when expressed such that compounds which disrupt E6/ERC55 or E6/E6bp
interactions confer positive growth selection to the cells.
EXAMPLES
Example 1: Sample Preparation
The E6bp peptide was synthesized using standard protein synthesis methods and
purified by HPLC. The recombinant E6 protein is expressed in E. coli,
solubilized from
inclusion bodies, and refolded using a rapid dilution method. The E6 protein
has been
expressed and solubilized from HPV-16 to a concentration of 4mg/mL (0.2 mM).
For
the peptide-E6 complex, (protonated) peptide is added to 0.2 mM deuterated
(2H)E6
following the procedure of Shibata et al., ( 1995) Arch. Biochem. Biophys.
319:204, the
contents of which are incorporated herein by reference. This allows
observation of only
the 1 H NMR resonances of the E6-bound peptides.
Example 2: NMR Spectroscopy
Peptide samples were prepared at about 2 mM concentration. One- and two-
dimensional (1D & 2D) NMR data were collected on a S00 MHz Bruker AMX500
spectrometer. E6bp is titrated with calcium and NMR spectra recorded to
determine the
effect on the peptide conformation. Low concentration peptide samples (0.2 mM)
are
titrated with E6 to determine the residues that are affected most by E6.
Samples are
prepared in both H20 and D20 solution.
Since the equilibrium binding constant of E6bp-E6 complex is in the p,M range,
transferred NOE (TRNOE) experiments, as described in Clore M. and Gronenborn
A.M., ( 1982) J. Magn. Reson. 48:402; ibid. ( 1983) 53:423; and Sykes B.D., (
1994) Cur.
Opin. Biotech. 4:392, the contents of which are incorporated herein by
reference, are
suitable to determine the conformation of E6-bound E6bp. TRNOE experiments are
performed with an approximately 10-fold excess of ligand relative to the
protein (2 mM
peptide and 0.2 mM E6 protein). In TRNOE experiments, the spectra exhibit
mostly the
lineshape and intensity of the unbound peptide, but the NOESY cross peaks
represent
the bound conformation of the peptide. Spectra are optimized in terms of salt
concentration (up to 0.2 M) and temperatures (5°C to 35°C) to
identify the conditions
CA 02273186 1999-06-02
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that yield superior quality NMR data with narrow line shapes, following the
methods
described in Baleja J.D., (1996) Techniques in Protein Chemistry VII:131, the
contents
of which are incorporated herein by reference.
Total correlation spectroscopy (TOCSY), and nuclear Overhauser effect
spectroscopy (NOESY) experiments are used for resonance assignment and to
obtain
conformation data for structure calculations, as described in Bax A. and Davis
D.G.,
( 1985) J. Magn. Reson. 65:355; and Wuthrich K., ( 1986) NMR of Proteins and
Nucleic
Acids, Wiley, New York, the contents of which are incorporated herein by
reference.
NMR data are processed using the Bruker NMR processing program or FELIX
(Biosym,
Inc.)
Example 3: NMR Resonance Assignment Strategy
NMR resonance strategy is performed as described in Wiithrich et al. In this
method, TOCSY spectra are analyzed to provide information regarding spin-spin
coupled NMR-active nuclei. Different types of amino acids side chains produce
distinct
TOCSY cross peak patterns. The cross-peak patterns for each amino acid are
linked
together using the NOESY experiment since NOE sequential connectivities can be
identified between residues neighboring in sequence. NOESY spectra show cross
peaks
between protons that are within 5 t~ of each other and therefore also show
contacts
between residues that are sequentially distant but spatially close.
Interproton distances
are determined from NOESY cross peak intensities. ~ and x-1 torsion angles are
obtained by measuring the coupling constants from 1 D slices of resolution-
enhanced 2D
data, as described in Wiithrich K., (1986) NMR ofProteins and Nucleic Acids,
Wiley,
New York; and Szyperski T. et al., (1992) J. Magn. Reson. 99:552, the contents
of
which are incorporated herein by reference.
~ H NMR resonance assignments were made (see Table II) and a low resolution
structure of the calcium-bound E6bp was calculated using approximately 200
interproton distances and 15 ~ torsion angles derived from NOESY data and
distance
geometry and simulated annealing protocols of the INSIGHTII molecular modeling
program (see Figure 1 ). Consistent with peptide analogs of other EF-hand
proteins, the
E6bp peptide dimerizes. From the dispersion present in the NMR spectra, it is
estimated
that 400 additional interproton distances are obtainable. Structure
calculation is repeated
with the more extensive data set to determine the high resolution structure of
E6bp. The
high resolution structure of E6bp both free and bound to E6 protein is also
solved.
Knowing the conformation for this E6bp in the absence of E6 protein is
important for
understanding the conformational changes brought about by binding to E6 and
the
binding surface of the peptide involved in the interaction.
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TABLE II
NMR Resonance Assignments for E6 binding peptide
Residue HN HA HB HG Others
Glu 1 - 4.02 2.08,2.08 2.37,2.37
Phe 2 8.10 4.76 3.08,3.03 D, 7.21; E, 7.15;
Z, 7.28*
Val3 8.11 4.16 1.95 0.89,0.89
Ile 4 8.11 3.98 1.79 1.46) G2, 0.86 D 1, 0.80
1.12
Gln ~ 8.35 4.15 2.10,2.062.37,2.37E, 7.51,6.76
Glu 6 8.00 4.22 2.08,2.02*2.29,2.29
Ala 7 7.98 4.35 1.46
Leu 8 8.12 4.17 1.69,1.621.69 D) 0.81,0.79
Glu 9 8.22 3.98 2.08,2.072.36,2.34
Glu 10 7.98 4.11 1.87,1.842.12,2.12
His 11 8.03 4.63 3.33,3.12 D. 7.38; E, 8.30
Asp 12 7.73 4.76 3.00,2.37
Lys 13 7.95 4.15 1.97,1.921.64,1.56D, 1.70,1.70: E,
3.09.3.09
Asn 14 8.17 4.83 3.31,2.91 D, 7.97,6.75
Gly 15 7.66 3.92,3.84
Asp 16 8.06 4.56 3.17,2.53
Gly 17 9.93 4.10,3.48
Phe 18 8.05 4.90 2.95,2.88 D, 7.12; E, 7.26;
Z, 7.38*
Va119 8.78 4.70 1.96 0.93,0.83
Ser 20 9.11 4.72 4.49,4.05
Leu 21 8.77 3.99 1.67,1.521.50* D, 0.73,0.71
Glu 22 8.85 3.92 2.07,1.982.37,2.29
Glu 23 7.79 4.05 2.70,2.552.62,2.33
Phe 24 8.30 4.08 3.03,2.95 D, 6.72; E, 6.88;
Z,6.89*
Leu 25 8.56 4.03 I .78,1.501.50 D, 0.72,0.69
Gly 26 7.78 3.92,3.92*
Asp 27 7.71 4.74 2.70,2.55
Tyr 28 7.73 4.32 2.78,2.70 D, 6.80; E, 6.62
Arg 29 7.67 4.25 1.64,1.541.38 D) 3.04,3.04; E,
7.31, H, -
Trp 30 7.88 4.60 3.32,3.18 D) 7.25; E1, 10.01;
E3, 7.58
Z2, 7.35; Z3, 7.08;
H, 7.08
Asp 31 8.09 4.53 2.55,2.44
NH2 7.56* 6.86*.
* Tentative assignment. "-" not observable. 1 mM peptide was in a buffer
containing
100 mM NaCI, 50 mM calcium chloride, pH 6.0, 10% TFE, 35 deg. C.
Structure Calculation and Analysis
The interproton distances and torsion angle information (see Table III) are
introduced into the distance geometry program, DGII of Insight (Biosym, Inc.).
The
initial structure calculated using distance geometry and simulated annealing
protocols is
refined by determining the presence or lack of NOE cross peaks for spatially
proximal
protons in the calculated structure.
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Structures are compared to one another to highlight the conformational
changes.
For example, the E6bp peptide is expected to have major changes in surface
properties
on binding calcium, such as the formation of a deep hydrophabic cavity
essential for
target protein recognition (see Ikura M., ( 1996) Trends Biochem. Sci. 21:14,
the contents
of which are incorporated herein by reference). The structures of E6ap and
E6bp are
compared to one another to test the hypothesis that they are similar. The
residues
involved in interaction with E6 are identified by chemical shift changes that
occur upon
titration, as described in Baleja J.D. et al., (1992) Nature 356:450; and
Baleja J.D. et al.,
( 1992) Biochemistry 33:3071, the contents of which are incorporated herein by
reference. Furthermore, differences in conformation observed on binding E6 are
determined.
The high resolution structures and the identification of key residues for
binding
E6 are important for the design of small molecule inhibitors that block the
oncogenic
properties of E6 ( see Fesik S. W., ( 1993) J. Biomol. NMR 3:261; and Kuntz
LD. et al.,
( 1994) Acc. Chem. Res. 27:117, the contents of which are incorporated herein
by
reference). The first focus is on readily synthesized peptide derivatives for
in vitro
testing. Modifications of a lead compound to optimize interactions are
proposed using
the BUILDER and LUDI features of the INSIGHTII molecular modeling program (and
chemical intuition). Binding to the protein surface uses the DOCK module. An
illustration of the approach is given in Figure 3.
TABLE III
STRUCTURE DATA FOR E6 BINDING PEPTIDE
BIOSYM restraint 1
remote nrochiral center
VAL 19:HG1*VAL 19:HG2*~ VAL 19:CG1VAL_19:CG2 VAL 19:CB
VAL 19B:HG1*VAL_19B:HG2*VAL 19B:CG1VAL 19B:CG2VAL_19B:CB
LEU_21:HD1*LEU_21:HD2*LEU_21:CD1 LEU_21:CD2 LEU_21:CG
LEU 21B:HD1*LEU 21B:HD2*LEU 21B:CD1LEU 21B:CD2LEU 21B:CG
NOE distance
Distance between atoms (interproton distances) within the E6 binding peptide
ASP- 12:OD1CAL_40:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
ASP-_12B:OD1CAL_40B:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
ASN 14:OD1 CAL 40:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
ASN l4B:OD1CAL 40B:CAG2.000 2.?20 2.800 1.00 1.001000.0000.00
ASP- IG:ODICAL 40:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
ASP- 1GB:OD1CAL_40B:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
GLU- 23:OE1CAL 40:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
GLU- 23B:OE1CAL_40B:CAG2.000 2.720 2.800 1.00 1.001000.0000.00
GLU- 23:OE2CAL 40:CA62.000 2.720 2.800 1.00 1.001000.0000.00
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GLU- 23B:OE2CAL_40B:CA62.000 2.720 2.8001.00 1.00 1000.0000.00
PHE 18:0 CAL_40:CA62.000 2.720 2.8001.00 1.00 1000.0000.00
PHE 18B:0 CAL_40B:CA62.000 2.720 2.8001.00 1.00 1000.0000.00
ILE 4:HN GLN_S:HN 2.000 4.000 2.8001.00 1.00 1000.0000.00
ILE 4B:HN GLN SB:HN 2.000 4.000 2.8001.00 1.00 1000.0000.00
ILE 4:HB GLN_S:HN 2.000 4.000 2.8001.00 1.00 1000.0000.00
1LE 4B:HB GLN SB:HN 2.000 4.000 2.8001.00 1.00 1000.0000.00
GLN S:HN GLU- 6:HN 2.000 4.000 2.8001.00 1.00 1000.0000.00
GLN_SB:HN GLU- 6B:HN2.000 4.000 2.8001.00 1.00 1000.0000.00
GLN S:HB* GLU- 6:HN 2.000 4.500 2.8001.00 1.00 1000.0000.00
GLN SB:HB* GLU- GB:HN2000 4.500 2.8001.00 1.00 1000.0000.00
GLN_S:HG* GLU- 6:HN 2.OJ0 6.000 2.8001.00 1.00 1000.0000.00
GLN SB:HG* GLU- 6B:HN2.000 6.000 2.8001.00 1.00 1000.0000.00
GLU- G:HN ALA 7:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
GLU- GB:HN ALA_7B:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
GLU- G:HG* ALA 7:HN 2.000 6.000 2.8001.00 1.00 1000.0000.00
GLU- GB:HG*ALA_7B:HN 2.000 6.000 2.8001.00 1.00 1000.0000.00
ALA 7:HN LEU 8:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
ALA 7B:HN LEU 8B:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
ALA 7:HB* LEU 8:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
ALA_7B:HB* LEU 8B:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
LEU 8:HN GLU-_9:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
LEU 8B:HN GLU-_9B:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
GLU- 9:HN GLU- IO:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
GLU- 9B:HN GLU-_IOB:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
HIS lI:HN ASP-_12:HN2.000 4.000 2.8001.OU 1.00 1000.0000.00
HIS_I1B:HN ASP-_12B:HN2.000 4.000 2.8001.00 1.00 1000.0000.00
ASP- 12:HN LYS+ 13:HN2.000 4.000 2.8001.00 1.00 1000.0000.00
ASP- 12B:HNLYS+ 13B:HN2.000 4.000 2.8001.00 1.00 1000.0000.00
LYS+ 13:HN ASN_14:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
LYS+ 13B:HNASN 14B:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
ASN 14:HN GLY 15:HN 2.000 3.000 2.8001.00 1.00 1000.0000.00
ASN 14B:HN GLY 15B:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
ASN_14:HN GLY_15:HAI2.000 6.000 2.8001.00 1.00 1000.0000.00
ASN 14B:HN GLY 15B:HA12.000 6.000 2.8001.00 1.00 1000.0000.00
ASN 14:HN GLY 15:HA22.000 6.000 2.8001.00 1.00 1000.0000.00
ASN 14B:HN GLY_15B:HA22.000 6.000 2.8001.00 1.00 1000.0000.00
GLY_15:HN ASP-_16:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
GLY 15B:HN ASP-_iGB:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
GLY I S:HA ASP- 1 2.000 3.500 2.8001.00 1.00 1000.0000.00
i G:HN
GLY 15:HA2 ASP-_1G:HN2.000 3.500 2.8001.00 1.00 1000.0000.00
GLY 158:HA1ASP-_IGB:HN2.000 3.500 2.8001.00 1.00 1000.0000.00
GLY ISB:HA2ASP-_16B:HN2.000 3.500 2.8001.00 1.00 1000.0000.00
ASP- 1G:HN GLY_17:HN 2.000 2.900 2.8001.00 1.00 1000.0000.00
ASP- 1GB:HNGLY_17B:HN2.000 2.900 2.8001.00 1.00 1000.0000.00
ASP- 16:HA GLY_17:HN 3.000 5.000 2.8001.00 1.00 1000.0000.00
ASP-_1GB:HAGLY_17B:HN3.000 5.000 2.8001.00 1.00 1000.0000.00
GLY 17:HA1 PHE 18:HN 2.000 2.500 2.8001.00 1.00 1000.0000.00
GLY 17:HA2 PHE_I8:HN 2.000 2.500 2.8001.00 1.00 1000.0000.00
GLY 17B:HA PHE I8B:HN2.000 2.500 2.8001.00 1.00 1000.0000.00
1
GLY_17B:HA2PHE_18B:HN2.000 2.500 2.8001.00 1.00 1000.0000.00
PHE I8:HA VAL_19:HN 2.000 2.500 2.8001.00 1.00 1000.0000.00
PHE 18B:HA VAL_19B:HN2.000 2.500 2.8001.00 1.00 1000.0000.00
LEU 21:HN GLU- 22:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
LEU 21B:HN GLU- 22B:HN2.000 3.000 2.8001.00 1.00 1000.0000.00
LEU_21:HB* GLU-_22:HN2.000 4.500 2.8001.00 1.00 1000.0000.00
LEU 218:HB*GLU- 22B:HN2.000 4.500 2.8001.00 1.00 1000.0000.00
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GLU-_22:HN GLU- 23:HN2.000 3.000 2.800 1.00 1.001000.000U.00
~
GLU- 22B:HN 23B:HN 2.000 3.000 2.800 1.00 1.001000.0000.00
GLU-
GLU-_22:HB* GLU- 23:HN2.000 4.500 2.800 1.00 1.001000.0000.00
GLU-_22B:HB*GLU- 23B:HN2.000 4.500 2.800 1.00 1.001000.0000.00
GLU-_23:HN PHE_24:HN 2.000 3.000 2.800 1.00 1.001000.0000.00
GLU- 23B:HN PHE_24B:HN2.000 3.000 2.800 1.00 1.001000.0000.00
GLU-_23:HB* PHE_24:HN 2.000 4.500 2.800 1.00 1.001000.0000.00
GLU-_23B:HB*PHE_24B:HN2.000 4.500 2.800 1.00 1.001000.0000.00
PHE_24:HN LEU 25:HN 2.000 3.000 2.800 1.00 1.001000.0000.00
PHE 24B:HN LEU 25B:HN2.000 3.000 2.800 1.OU 1.001000.0000.00
PHE 24:HB' LEU_25:HN 2.000 4.500 2.800 '.~.001.001000.0000.00
'
_24B:HB' LEU 25B:HN2.000 4.500 2.800 1.00 1.001000.0000.00
PHE
LEU 25:HN GLY_2G:HN 2.000 3.000 2.800 1.00 1.001000.0000.00
LEU 25B:HN GLY 26B:HN2.000 3.000 2.800 1.00 1.001000.0000.00
LEU_25:HB' GLY 2G:HN 2.000 4.500 2.800 1.00 1.001000.0000.00
LEU 25B:HB' GLY 2GB:HN2.000 4.500 2.800 1.00 1.001000.0000.00
GLY_26:HN ASP- 27:HN2.000 3.500 2.800 I.OU 1.001000.0000.00
GLY 26B:HN ASP- 278:HN2.000 3.500 2.800 1.00 1.001000.0000.00
ASP- 27:HN TYR 28:HN 2.000 3.500 2.800 1.00 1.001000.0000.00
ASP- 27B:HN TYR 28B:HN2.000 3.500 2.800 1.00 1.001000.0000.00
TYR 28:HN ARG+_29:HN2.000 4.000 2.800 1.00 1.001000.0000.00
TYR 28B:HN ARG+_29B:HN2.000 4.000 2.800 1.00 1.001000.0000.00
TYR 28:HB' ARG+ 29:HN2.000 4.500 2.800 1.00 1.001000.0000.00
TYR_28B:HB' ARG+ 29B:HN2.000 4.500 2.800 1.00 1.001000.0000.00
ARG+ 29:HN TRP_30:HN 2.000 5.000 2.800 1.00 1.001000.0000.00
ARG+ 29B:HN TRP_30B:HN2.000 5.000 2.800 1.00 1.001000.0000.00
ARG+ 29:HB' TRP_30:HN 3.000 6.000 2.800 1.00 1.001000.0000.00
ARG+ 29B:HB*TRP 30B:HN3.000 6.000 2.800 1.00 1.001000.0000.00
ARG+ 29:HG* TRP 30:HN 3.000 6.000 2.800 f.00 1.001000.0000.00
ARG+ 29B:HG'TRP 30B:HN3.000 6.000 2.800 1.00 1.001000.0000.00
ARG+ 29:HA TRP 30:HN 2.000 2.600 2.800 1.00 1.001000.0000.00
ARG+ 29B:HA TRP 30B:HN2.000 2.600 2.800 1.00 1.001000.0000.00
TRP 30:HN AP-C 31:HN2.000 5.000 2.800 1.00 1.001000.0000.00
TRP 30B:HN AP-C 31B:HN2.000 5.000 2.800 1.00 1.001000.0000.00
ILE 4:HA ALA_7:HB' 2.000 4.000 2.800 1.00 1.001000.0000.00
ILE_4B:HA ALA_7B:HB'2.000 4.000 2.800 1.00 1.001000.0000.00
GLN $:HN ALA_7:HB* 2.000 5.000 2.800 1.00 1.001000.0000.00
GLN $B:HN ALA_7B:HB*2.000 5.000 2.800 1.00 1.001000.0000.00
ALA 7:HA GLU-_IO:HB*2.000 4.000 2.800 1.00 1.001000.0000.00
ALA 7B:HA GLU- IOB:HB*2.000 4.000 2.800 1.00 1.001000.0000.00
ALA 7:HA HIS II:HD22.000 5.000 2.800 1.00 1.001000.0000.00
ALA 7B:HA HIS I1B:HD22.000 5.000 2.800 1.00 1.0U1000.0000.00
ALA 7:HB' GLU-_9:HN 2.000 5.000 2.800 1.00 1.001000.0000.00
ALA 7B:HB' GLU-_9B:HN2.000 5.000 2.800 1.00 1.0U1000.0000.00
ALA 7:HB' GLU-_IO:HG*2.000 8.000 2.800 1.00 1.001000.0000.00
ALA 7B:HB* GLU-_IOB:HG*2.000 8.000 2.800 1.00 1.001000.0000.00
GLU- 9:HA HIS 11:HN 2.000 5.000 2.800 1.00 1.001000.0000.00
GLU-_9B:HA H1S_11B:HN2.000 5.000 2.800 1.00 1.001000.0000.00
HIS_I1:HB' VAL_19:HG1*2.000 7.000 2.800 1.00 1.001000.0000.00
HIS I1B:HB' VAL 19B:HG1'2.000 7.000 2.800 1.00 1.001000.0000.00
HIS 1HB' VAL_19:HG2'2.000 7.000 2.800 1.00 1.001000.0000.00
HIS 1 1 B:HB'VAL 198:HG2*2.000 7.000 2.800 1.00 1.001000.0000.00
ASN 14:HD21 ASP- 1G:HB'2.000 7.000 2.800 1.00 1.001000.0000.00
ASN 14B:HD21ASP-_IGB:HB'2.000 7.000 2.800 1.00 1.001000.000O.UO
GLY_i5:HA1 GLY_17:HN 2.000 5.000 2.800 1.00 1.001000.0000.00
GLY 1S:HA2 GLY_17:HN 2.000 5.000 2.800 I.OU 1.001000.0000.00
GLY lSB:HA GLY 17B:HN2.000 5.000 2.800 1.00 1.001000.0000.00
I
*rB
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GLY 15B:HA2GLY_17B:HN 2.000 5.000 2.800 1.001.00 1000.0000.00
SER 20:HN GLU- 23:HN 2.000 5.900 2.800 1.001.00 1000.0000.00
SER 20B:HN GLU-_23B:HN2.000 5.900 2.800 1.001.00 1000.0000.00
SER 20:HN GLU- 23:HB*2.000 5.900 2.800 1.001.00 1000.0000.00
SER 20B:HN GLU-_23B:HB'2.000 5.900 2.800 1.001.00 1000.0000.00
SER 20:HN GLU-_23:HG'2.000 6.900 2.800 1.001.00 1000.0000.00
SER 20B:HN GLU- 23B:HG'2.000 6.900 2.800 1.001.00 1000.0000.00
LEU 21:HA PHE_24:HB* 2.000 4.000 2.800 1.001.00 1000.0000.00
LEU 21B:HA PHE_24B:HB*2.000 4.000 2.800 1.001.00 1000.0000.00
LEU 21:HD1 PHE_24:HB' 2.000 7.000 2.800 I 1.00 1000.0000.00
* .00
LEU 21B:HD1'PHE_24B:HB*2.000 7.000 2.800 1.001.00 1000.0000.00
LEU 21:HD2'PHE_24:HB* 2.000 7.000 2.800 1.001.00 1000.0000.00
LEU 21B:HD2*PHE_24B:HB*2.000 7.000 2.800 1.001.00 1000.0000.00
GLU-_22:HA PHE_24:HN 2.000 5.000 2.800 1.001.00 1000.0000.00
GLU-_22B:HAPHE_24B:HN 2.000 5.000 2.800 1.001.00 1000.0000.00
GLU- 22:HA LEU_25:HN 2.000 4.000 2.800 1.001.00 1000.0000.00
GLU- 22B:HALEU 25B:HN 2.000 4.000 2.800 1.001.00 1000.0000.00
GLU-_22:HA LEU_25:HB* 2.000 4.000 2.800 1.001.00 1000.0000.00
GLU-_22B:HALEU 25B:HB'2.000 4.000 2.800 1.00t.00 1000.0000.00
PHE 24:HA ASP- 27:HB*2.000 4.000 2.800 1.001.00 1000.0000.00
PHE 24B:HA ASP-_27B:HB'2.000 4.000 2.800 t.001.00 1000.0000.00
PHE 24:HB' GLY_26:HN 2.000 6.000 2.800 1.001.00 1000.0000.00
PHE 24B:HB'GLY_26B:HN 2.000 6.000 2.800 1.001.00 1000.0000.00
LEU 25:HA TYR_28:HB* 2.000 4.300 2.800 1.001.00 1000.0000.00
LEU 25B:HA TYR_28B:HB*2.000 4.300 2.800 1.001.00 1000.0000.00
ALA_7:HA PHE 24B:CG 3.500 99.0002.800 1.001.00 1000.0000.00
ALA 7B:HA PHE_24:CG 3.500 99.0002.800 1.001.00 1000.0000.00
ALA 7:HA PHE_24B:CZ 3.500 99.0002.800 1.001.00 1000.0000.00
ALA_7B:HA PHE_24:CZ 3.500 99.0002.800 1.001.00 1000.0000.00
ALA 7:HB* PHE_24B:CG 2.000 7.000 2.800 1.001.00 1000.0000.00
ALA_7B:HB* PHE_24:CG 2.000 7.000 2.800 1.001.00 1000.0000.00
ALA 7:HB* PHE_24B:CZ 2.000 7.000 2.800 1.001.00 1000.0000.00
ALA_7B:HB* PHE_24:CZ 2.000 7.000 2.800 1.001.00 1000.0000.00
1LE 4:HD1* TYR_28B:CG 2.000 7.000 2.800 1.001.00 1000.0000.00
ILE 4B:HD1'TYR 28:CG 2.000 7.000 2.800 1.001.00 1000.0000.00
ILE 4:HDl* TYR 28B:CZ 2.000 7.000 2.800 1.001.00 1000.0000.00
ILE 4B:HD1*TYR_28:CZ 2.000 7.000 2.800 1.001.00 1000.0000.00
LEU_8:CG PHE 24B:CG 2.000 9.000 2.800 1.001.00 1000.0000.00
LEU_8B:CG PHE 24:CG 2.000 9.000 2.800 1.001.00 1000.0000.00
PHE_I8:CG SER 20B:HB'2.000 9.000 2.800 1.001.00 1000.0000.00
PHE_18B:CG SER_20:HB' 2.000 9.000 2.800 1.001.00 1000.0000.00
PHE 18:HN LEU 21B:HB*3.500 99.0002.800 1.001.00 1000.0000.00
PHE 18B:HN LEU 21:HB* 3.500 99.0002.800 1.001.00 1000.0000.00
PHE 18:HA SER 20B:HA 2.000 3.000 2.800 1.001.00 1000.0000.00
PHE 18B:HA SER 20:HA 2.000 3.000 2.800 1.001.00 1000.0000.00
33 dihedral
torsion ankle information (theta and chi-1)
LEU 21:HN LEU 21:N LEU_21:CA LEU 21:HA5.5 I.0G0.0G0.01000.0-145.6
GLU- 22:HNGLU- 22:NGLU-_22:CAGLU- 22:HA5.5 I.0G0.0G0.01000.0-145.6
PHE 24:HN PHE 24:N PHE 24:CA PHE 24:HA5.5 1.UG0.0G0.01000.0-(45.G
LEU 25:HN LEU 25:N LEU 25:CA LEU 25:HA5.5 I.0G0.0G0.01000.0-145.6
LEU_25:C GLY 2G:N GLY_26:CA GLY_2G:C 5.5 1.0G0.0G0.01000.0-90.0
ASP- 27:HNASP- 27:NASP-_27:CAASP- 27:HA5.5 I.0G0.0G0.01000.0-145.6
TYR 28:HN TYR 28:N TYR 28:CA TYR 28:HA5.5 I.0G0.0G0.01000.0-ISS.G
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ARG+ 29:HNARG+_29:NARG+_29:CAARG+ 29:HAS.S 1.060.06U.0 1000.0-155.6
HIS_11:HN HIS_11:N HIS 11:CA HIS 11:HA5.5 1.060.060.0 1000.0-145.6
ASP- 12:HNASP-_12:NASP- 12:CAASP-_12:HAS.S 1.060.060.0 1000.0-145.6
LYS+ 13:HNLYS+ 13:NLYS+ 13:CALYS+ 13:HAS.S 1.060.0G0.0 1000.0-145.6
ASN 14:HN ASN_14:N ASN_14:CA ASN_14:HAS.S 1.060.060.0 1000.0-14S.G
ASN_14:C GLY_IS:N GLY 1S:CA GLY iS:C S.S !.0G0.0G0.0 1000.0-90.0
ASP- 16:HNASP-_16:NASP-_16:CAASP-_iG:HAS.S I.060.0G0.0 1000.0-145.6
LEU 21B:HNLEU_21B:NLEU_21B:CALEU 21B:HAS.S 1.060.0G0.0 1000.0-14S.G
GLU- 22B:HNGLU-_22B:NGLU-_22B:CAGLU- 22B:HAS.S 1.0G0.0G0.0 1000.0-14S.G
PHE 24B:HNPHE_24B:NPHE 24B:CAPHE_24B:HAS.S 1.060.060.0 1000.0-14S.G
LEU 25B:HNLU_2SB:N LEU 25B:CALEU 25B:HAS.S 1.060.0G0.0 1000.0-145.6
LEU_25B:C GLY_26B:NGLY_26B:CAGLY_2GB:CS.S I.060.0G0.0 1000.0-90.0
ASP-_27B:HNASP-_27B:NASP-_27B:CAASP- 27B:HAS.S 1.0G0.0G0.0 1000.0-145.6
TYR_28B:HNTYR_28B:NTYR 28B:CATYR 28B:HAS.S I 60.060.0 1000.0-1
.0 SS.G
ARG+ 29B:HNARG+ 29B:NARG+ 29B:CAARG+ 29B:HAS.S 1.0G0.0G0.0 1000.0-I
SS.G
HIS 11B:HNHIS_11B:NHIS 11B:CAHIS_11B:HAS.S 1.0G0.0G0.0 1000.0-14S.G
ASP- 12B:HNASP-_12B:NASP- 12B:CAASP- 12B:HAS.S 1.0G0.0G0.0 1000.0-145.6
LYS+ 13B:HNLYS+ 13B:NLYS+ 13B:CALYS+ l3B:HAS.S 1.060.0G0.0 1000.0-145.6
ASN_14B:HNASN 14B:NASN_14B:CAASN l4B:HAS.S l.0G0.0G0.0 1000.0-14S.G
f
ASN_14B:C _ISB:N GLY_15B:CAGLY_ISB:CS.S 1.060.0G0.0 1000.0-90.0
GLY
ASP- 1 ASP- l6B:NASP- 16B:CAASP- 1 5.5 1.060.0G0.0 1000.0-14S.G
GB:HN GB:HA
Structure Analysis of ERC55
Sequence analysis of the E6-interacting protein, ERCSS, revealed homology to
proteins containing the calcium-binding helix-loop-helix motif called an EF
hands. Six
EF hands are predicted to occur in its C-terminal domain. Only one of these EF
hands, a
2S amino acid segment, E6bp, binds E6 selectively (Chen J.J. et al., ( 1995)
Science
269:529), and with about the same affinity as the full-length ERCSS protein.
Structural
determination of other EF-hand domains using IH NMR spectroscopy (Ikura M.
(1996)
Trends Biochem. Sci. 21:14) suggests the feasibility for NMR study of this
peptide. A
small amount of E6bp peptide was synthesized, purified and a 0.7 mM sample was
prepared. As predicted, the peptide bound calcium and showed excellent NMR
spectral
dispersion as shown in Figure 2. The calcium free form of E6bp was studied.
Structure Analysis of E6AP
An 18 amino acid residue peptide fragment, E6ap, is the minimal region of
1 S E6AP that binds E6. The sequence of E6ap is homologous to E6bp (see Table
I), and
their solution properties are likely to be similar. Sequence prediction
(Wishart et al.,
( 1994) Comp. Biol. Sci. 10:121 ) has indicated a helical structure for the
homologous
region of E6ap-the same region which has been determined to be a helical in
the
primary solution structure of E6bp (Figure 1). E6ap does not have the calcium
ligands
required for an EF hand, and therefore is unlikely to bind calcium. The small
sizes of
the E6bp and E6ap peptides make them attractive for detailed structural
studies by
solution NMR methods in a timely manner.
Example 4: Analysis of The E6bp Structure By Site-Directed Mutagenesis
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- 28 -
To further map the domain that is important for E6 binding, additional
deletion
mutants from E6bp were constructed as GST fasions and tested for binding with
E6.
Deletion of the first alpha helix (20mer) did not affect binding, nor did
deletion of two
additional amino acid residues from the C-terminal end of E6bp to the distal
region of
the second alpha helix affect binding. A 13 amino acid peptide containing the
second
alpha helix as a GST fusion retained the ability to bind to E6, although at
reduced
efficiency in comparison to intact E6bp. Notably, the major portion of the
loop region
from the EP-hand motif is deleted in the 13 amino acid peptide. The ability of
the
second alpha helix to bind E6 demonstrates that the interaction of E6BP with
E6 is
independent of calcium binding, as the first alpha helix and loop region from
the EP-
hand motif are both required for calcium binding.
Alanine replacement mutations in E6bp were also constructed and used to define
the amino acids) important for E6 interaction in the alpha helix. Some
mutations were
also made in the surrounding regions. As expected, mutants V 19A, S20A, E22A,
R29A,
and W30A, which have mutations in the area beyond the alpha helix, bound E6 at
wild-
type level (see Figure 4). Mutant F 18A showed some reduced binding. Notably,
phenyalanine is a hydrophobic residue, which may enhance the interaction
between E6
and E6bp. This may explain why a leucine to alanine change at amino acid 21
also
reduced binding. While mutants at the boundary of alpha helix (E23 and D28A)
showed
modest reduction (approximately 60% of wild-type binding) in their E6 binding
ability,
all other mutants made up of the alpha helical structure showed substantial
decrease in
E6 binding. Notably, a change of leucine to alanine at amino acid 25 totally
abolished
binding. The ability of mutants F 18A and E23A to bind E6 confirmed the notion
that
the interaction of E6BP with E6 is independent of calcium binding, as both
mutations
abolished calcium binding. Finally, a leucine to proline change at amino acid
25 of
E6bp was created and used in a binding experiment. This change (L25P) which is
expected to disrupt the alpha helical structure, totally abolished E6bp-
binding with E6,
indicating that the alpha helix from the conserved motif is indeed important
for E6
binding.
EQUIVALENTS
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
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1
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: James D. Baleja, Elliot J.Androphy and
Jason J.Chen
(ii) TITLE OF INVENTION:Structure-based Rational Design
of
Compounds to Inhibit Papillomavirus Infection
(iii) NUMBER OF SEQUENCES: 22
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: FISH & RICHARDSON
1S (B) STREET: 225 Franklin Street
(C) CITY: Boston
(D) STATE: Massachusetts
(E) COUNTRY: USA
(F) ZIP: 02110
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
3O (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/061,295
(B) FILING DATE: 07 October 1997
(viii)ATTORNEY/AGENT INFORMATION:
(A) NAME: Paul Louis Myers
(B) REGISTRATION NUMBER: 35,965
(C) REFERENCE/DOCKET NUMBER: NEP-006PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617)542-5070
(B) TELEFAX: (617)542-8906
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
$0 (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
SS (v) FRAGMENT TYPE: internal
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2
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Ile Pro Glu Ser Ser Glu Leu Thr Leu Gln Glu Leu Leu Gly Glu Glu
1 5 10 15
Arg Arg
IO (2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino
acids
(B) TYPE: amino acid
1$ (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
20
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:2:
Ala Leu Glu Glu His Asp Gly Asp Gly Phe Val Ser
Lys Asn Leu Glu
1 5 10 15
25
Glu Phe Leu Gly Asp Tyr Asp
Arg Trp
20 25
(2) INFORMATION
FOR
SEQ
ID N0:3:
30
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
35
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
4O (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:3:
Glu Leu Thr Leu Gln Glu Gly Glu Glu Arg
Leu Leu
1 5 10
4$ (2) INFORMATION
FOR
SEQ
ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino
acids
(B) TYPE: amino acid
$0 (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
$$
*rB
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(xi) SEQUENCE DESCRIPTION: N0:4:
SEQ ID
Met Asp Asp Leu Asp Ala
Leu Leu Ala Asp Leu
Glu Ser Thr
1 5 10
(2) INFORMATION
FOR
SEQ
ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
1$ (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: N0:5:
SEQ ID
Leu Gln Thr Leu Gln Asp Gly Asp Pro Gly Asp
Ile Leu Lys
1 5 to
(2) INFORMATION
FOR
SEQ
ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
2$ (A) LENGTH: 20 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:6:
3$ Asp Lys Asn Gly Asp Gly Ser Leu Glu Glu Phe Gly
Phe Val Leu Asp
1 5 10 15
Tyr Arg Trp Asp
20
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino
acids
4$ (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
$0 (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:7:
Asp Lys Ser Leu Glu Glu Phe Gly
Asn Leu Asp
Gly
Asp
Gly
Phe
Val
$$ 1 5 to is
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4
Tyr Arg
S (2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
Val Ser Leu Glu Glu Phe Leu Gly Asp Tyr Arg Trp Asp
1 S 10
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Ala Val Ser Leu Glu
1 5 to is
Glu Phe Leu Gly Asp Tyr Arg Trp Asp
20 25
4O (2} INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Ala Ser Leu Glu
1 5 10 15
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Glu Phe Leu Gly Asp Tyr Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID NO:11:
S
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
1S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ala Leu Glu
1 5 10 15
Glu Phe Leu Gly Asp Tyr Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:12:
ZS (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
3S
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Ala Glu
1 5 10 15
Glu Phe Leu Gly Asp Tyr Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
4S (A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
SO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
SS Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Ala
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1 5 10 15
Glu Phe Leu Gly Asp Tyr Asp
Arg Trp
20 25
(2)
INFORMATION
FOR
SEQ
ID
N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
1S (v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:14:
Ala Leu Glu Glu His Asp Gly Asp Gly Val Ser Leu
Lys Asn Phe Glu
1 5 to is
Ala Phe Leu Gly Asp Tyr Asp
Arg Trp
20 25
2S (2)
INFORMATION
FOR
SEQ
ID
N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION:
SEQ ID N0:15:
Ala Leu Glu Glu His Asp Gly Asp Gly Val Ser Leu
Lys Asn Phe Glu
1 5 10 15
Glu Ala Leu Gly Asp Tyr Asp
Arg Trp
20 25
(2) RMATION FOR SEQ ID N0:16:
INFO
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino
acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
S0
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
SS (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:16:
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Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
Glu Phe Ala Gly Asp Tyr Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:17:
lO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
Glu Phe Pro Gly Asp Tyr Arg Trp Asp
20 2s
(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
Glu Phe Leu Ala Asp Tyr Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
5~ (B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
SS (v) FRAGMENT TYPE: internal
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
S 1 5 10 15
Glu Phe Leu Gly Ala Tyr Arg Trp Asp
20 25
lO (2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
IS (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:20:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
2S
Glu Phe Leu Gly Asp Ala Arg Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
3S
(ii) MOLECULE TYPE: peptide
(v) FRAGMENT TYPE: internal
4O (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
4S Glu Phe Leu Gly Asp Tyr Ala Trp Asp
20 25
(2) INFORMATION FOR SEQ ID N0:22:
SO (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
S5 (ii) MOLECULE TYPE: peptide
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(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
Ala Leu Glu Glu His Asp Lys Asn Gly Asp Gly Phe Val Ser Leu Glu
1 5 10 15
Glu Phe Leu Gly Asp Tyr Arg Ala Asp
1~ 20 25
