Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02373918 2002-02-28
I
Title: Polo Domain Structure
A portion of the disclosure of this patent document contains material that is
subject to
copyright protection. The copyright owner has no objection to the facsimile
reproduction by
anyone of the patent document or patent disclosure, as it appears in the
Patent and Trademark
Office patent file or records, but otherwise reserves all copyright rights
whatsoever.
FIELD OF THE INVENTION
The present invention relates to two-, three- or four-dimensional structures
of a polo
domain. In particular, the invention relates to a crystal comprising a polo
domain. The crystal
may be useful for modeling and/or synthesizing mimetics of a polo domain or
ligands that
associate with the polo domain. Such mimetics or ligands may be capable of
acting as
modulators of activity of polo family kinases, and they may be useful for
treating, inhibiting, or
preventing diseases modulated by such kinases.
BACKGROUND
2o The polo family kinases in mammals includes 4 genes; Plk, Prk/Fnk, Snk and
Sak. C.
elegans has three and Drosophila has 2 polo-related kinases, while S.
cerevisiae and S. pombe
have only one each, CdcS and Plol, respectively. The polo family members play
multiple roles
in M-phase progression and cytokinesis. Mutation of polo in Drosophila, plol
in pombe and
cdc5 in S. cerevisiae cause mitotic defects including monopolar spindles,
aberrant chromosome
segregation, and failure of cytokinesis. In S. cerevisiae, these mitotic
defects can be rescued by
the heterologous expression of mammalian Plk or Prk/Fnk, suggesting that the
polo family
kinases share evolutionary conserved functions. Biochemical studies indicate
that the metazoan
polo family kinase Plk may be a positive regulator of cyclin B/Cdkl prior to
anaphase, and
thereafter a negative regulator that silences Cdkl by promoting the
destruction of cyclin B. The
Xenopus homolog, Plxl, phosphorylates the amino-terminal domain of Cdc25c
phosphatase,
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which in turn removes phosphate from thr-14 and tyr-15 of Cdkl kinase, thereby
activating
cyclin B/Cdkl and entry-into-mitosis: In late mitosis, Plk phosphorylates
proteins of the
anaphase-promoting complex (APC) and stimulates APC-dependent proteolysis of
cyclin B.
Polo family kinases are also checkpoint targets as DNA damage suppresses their
activity, thereby
delaying exit-from-mitosis.
The polo family kinases localize to characteristic mitotic structures during
cell cycle
progression, presumably to promote the interaction of the enzymes with
specific mitotic
substrates and effectors. Plk, Prk/Fnk, CdcS, plot, polol and Sak localize to
centrosornes in
early M phase and to the cleavage furrow or mother bud neck during
cytokinesis. Mutational
to analyses of cdc5 and plk have demonstrated a requirement and sufficiency of
the polo box motifs
for sub-cellular localization. In addition, these studies have demonstrated a
requirement for
proper sub-cellular localization for overall polo family function.
Citation of documents herein is not intended as an admission that any of the
documents
cited herein is pertinent prior art, or an admission that the cited documents
is considered material
to the patentability of any of the claims of the present application. All
statements as to the date or
representation as to the contents of these documents is based on the
information available to the
applicant and does not constitute any admission as to the correctness of the
dates or contents of
these documents.
2o SUMMARY OF THE INVENTION
Applicants have solved the x-ray crystal structure of a polo domain. Solving
the crystal
structure has enabled the determination of structural features of the polo
domain that permit the
design of modulators of proteins comprising a polo domain. The crystal
structure also enables
the determination of structural features in molecules or ligands that interact
or associate with the
polo domain.
Knowledge of the conformation of the polo domain and binding pockets thereof
is of
significant utility in drug discovery. The association of natural substrates
and effectors with a
polo domain and binding pockets thereof may be the basis of many biological
mechanisms. The
3o associations may occur with all or any parts of a polo domain. An
understanding of the
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association of a drug with the polo domain or part thereof will lead to the
design and
optimization of drugs having favorable associations with target polo family
kinases and thus
provide improved biological effects. Therefore, information about the shape
and structure of the
polo domain is valuable in designing potential modulators of proteins
comprising a polo domain
for use in treating diseases and conditions associated with or modulated by
the proteins.
The present invention relates to a two-, three- or four dimensional structure
of a polo
domain, or a binding pocket thereof.
The invention also relates to a crystal comprising a polo domain.
In an embodiment, the invention provides a crystal comprising a polo domain or
binding
to pocket thereof.
The present invention also contemplates molecules or molecular complexes that
comprise
all or parts of either one or more a polo domain, or homologs thereof, that
have similar structure
and shape.
The present invention also provides a crystal comprising a polo domain or
binding pocket
thereof and at least one ligand. A ligand may be complexed or associated with
a polo domain or
binding pocket thereof. Ligands include a substrate or analogue thereof or
effector. A ligand may
be a modulator of the activity of a polo family kinase
In an aspect the invention contemplates a crystal comprising a polo domain or
binding
pocket thereof complexed with a ligand (e.g. substrate or analogue thereof)
from which it is
2o possible to derive structural data for the ligand (e.g. substrate or
analogue thereof).
The shape and structure of a polo domain or binding pocket thereof may be
defined by
selected atomic contacts in the domain or pocket. In an embodiment, the polo
domain binding
pocket is defined by one or more atomic interactions or enzyme atomic
contacts.
An isolated polypeptide comprising a polo domain or binding pocket thereof
with the
shape arid structure of a polo domain or binding pocket thereof described
herein is also within
the scope of the invention.
The invention also provides a method for preparing a crystal of the invention,
preferably
a crystal of a polo domain or binding pocket thereof, or a complex of such a
domain or binding
pocket thereof, and a ligand.
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Crystal structures of the invention enable a model to be produced for a polo
domain or
binding pocket thereof, or complexes or parts thereof. The models will provide
structural
information about a polo domain, or a ligand and its interactions with a polo
domain or binding
pocket thereof. Models may also be produced for ligands. A model and/or the
crystal structure of
the present invention may be stored on a computer-readable medium.
A crystal and/or model of the invention may be used in a method of determining
the
secondary and/or tertiary structures of a polypeptide or binding pocket
thereof with incompletely
characterised structure. Thus, a method is provided for determining at least a
portion of the
secondary and/or tertiary structure of molecules or molecular complexes which
contain at least
1o some structurally similar features to a polo domain or binding pocket
thereof of the invention.
This is achieved by using at least some of the structural coordinates set out
in 2.
A crystal of the invention may be useful for designing, modeling, identifying,
evaluating,
and/or synthesizing mimetics of a polo domain or binding pocket thereof, or
ligands that
associate with a binding pocket. Such mimetics or ligands may be capable of
acting as
is modulators of polo kinase activity, and they may be useful for treating,
inhibiting, or preventing
conditions or diseases modulated by such kinases.
Thus the present invention contemplates a method of identifying a modulator of
a polo
family kinase comprising the step of applying the structural coordinates of a
polo domain or
binding pocket thereof, or atomic interactions, or atomic contacts thereof, to
computationally
2o evaluate a test ligand for its ability to associate with the polo domain or
binding pocket thereof.
Use of the structural coordinates of a polo domain or binding pocket thereof,
or atomic
interactions, or atomic contacts thereof to design or identify a modulator is
also provided.
The invention further contemplates classes of modulators of polo family
kinases based on
the shape and structure of a ligand defined in relation to the molecule's
spatial association with a
25 polo domain or binding pocket thereof. Generally, a method is provided for
designing potential
inhibitors of polo family kinases comprising the step of applying the
structural coordinates of a
ligand defined in relation to its spatial association with a polo domain or
binding pocket thereof,
to generate a compound that is capable of associating with the polo domain or
binding pocket
thereof.
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It will be appreciated that a modulator of a polo family kinase may be
identified by
generating an actual secondary or three-dimensional model of a polo domain or
binding pocket
thereof, synthesizing a compound, and examining the components to find whether
the required
mteracrion occurs.
5 Therefore, the methods of the invention for identifying modulators may
comprise one or
more of the following additional steps:
(a) testing whether the modulator is a modulator of the activity of polo
family kinases,
preferably testing the activity of the modulator in cellular assays and animal
model
assays;
(b) modifying the modulator;
(c) optionally rerunning steps (a) or (b); and
(d) preparing a pharmaceutical composition comprising the modulator.
Steps (a), (b) (c) and (d) may be carried out in any order, at different
points in time, and
they need not be sequential.
A potential modulator of a polo family kinase identified by a method of the
present
invention may be confirmed as a modulator by synthesizing the compound, and
testing its effect
on the polo family kinase in an assay for enzymatic activity. Such assays are
known in the art
(e.g phosphorylation assays).
A modulator of the invention may be converted using customary methods into
2o pharmaceutical compositions. A modulator may be formulated into a
pharmaceutical
composition containing a modulator either alone or together with other active
substances.
The invention also contemplates a method of treating or preventing a disease
or condition
associated with polo family kinases in a cellular organism, comprising:
(a) administering a modulator of the invention in an acceptable pharmaceutical
preparation;
and
(b) activating or inhibiting the polo family kinases to treat or prevent the
disease or
condition.
The invention provides for the use of a modulator identified by the methods of
the
invention in the preparation of a medicament to treat or prevent a disease in
a cellular organism.
3o Use of modulators of the invention to manufacture a medicament is also
provided.
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Still another aspect of the present invention provides a method of conducting
a drug
discovery business comprising:
(a) providing one or more systems for identifying modulators based on the
structure of a
polo domain or binding pocket thereof;
(b) conducting therapeutic profiling of modulators identified in step (a), or
further
analogs thereof, for efficacy and toxicity in animals; and
(c) formulating a pharmaceutical preparation including one or more modulators
identified in step (b) as having an acceptable therapeutic profile.
In certain embodiments, the subject method can also include a step of
establishing a
distribution system for distributing the pharmaceutical preparation for sale,
and may optionally
include establishing a sales group for marketing the pharmaceutical
preparation.
Yet another aspect of the invention provides a method of conducting a target
discovery
business comprising:
(a) providing one or more systems for identifying modulators based on the
structure of a
polo domain or binding pocket thereof;
(b) (optionally) conducting therapeutic profiling of modulators identified in
step (a) for
efficacy and toxicity in animals; and
(c) licensing, to a third party, the rights for further drug development
and/or sales for
agents identified in step (a), or analogs thereof.
These arid other aspects of the present invention will become evident upon
reference to
the following detailed description and Tables, and attached drawings.
DESCRIPTION OF THE DRAWINGS AND TABLES
The present invention will now be described only by way of example, in which
reference
will be made to the following Figures:
Figure 1. Sequence alignment of the polo domains from various polo family
kinases. Sak
polo domain orthologues are shown on the top and polo domain one and two of
other polo family
kinases are shown in the middle and bottom respectively. Secondary structure
for the polo
domain of Sak is indicated above the alignment. Residue numbers for the start
of each polo
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domain are shown on the left. Conserved hydrophobic residues are colored
yellow, acidic
residues red, basic residues blue, amine residues orange, hydroxyl residues
purple, and glycines
are colored green. Conserved dimer interface positions (T) and interfacial
cleft and pocket
residues (~) are as indicated.
Figure 2. Static light scattering analysis indicates polo domain dimerization
in solution.
Black plotline indicates refractive index of protein solution, which is
correlated with protein
concentration (scale not shown). The mass of monomeric polo domain is about
llkD; the red
line relays an average sample mass of about 23kD (scale to the left).
Figure 3. Sak polo domain crystals. Crytal structure was determined from a MAD
data set
1o was collected on the crystals of hexagonal morphology shown in A.
Figure 4. Ribbon diagram of polo domain dimer. A. Frontal view. B. Underside,
facing
into the hydrophobic pocket. Ribbons depiction of the polo domain homo-dimer
viewed a) down
the twofold symmetry axis and b) perpendicular to the symmetry axis. The dimer
subunits are
coloured red and blue and a-helices are labeled. All ribbon diagrams were
generated using
RIBBONS [Carson, 1991].
Figure 5. Surface representation of Sak-a polo domain. A. Front view and B.
Underside,
facing the cleft region and entrance to the pocket. C. A slab view of the
front face reveals a
pocket that is largely hydrophobic in nature. Squares: Hydrophobic residues.
The molecular
surface representation of of the poli domain homodimer. The molecular surface
of one subunit is
shown with hydrophobic (Met, Val, Leu, Ile, Phe,) residues coloured in green.
The two
perspectives differ by a 90° rotation about the Horizontal axis. In
panel b the twofold rotation
axis relating the two subunits of the dimer is shown. The buried surface area
of the dimer
interface is 2447.78 A2. Panel 5c illustrates the approximate dimensions of he
internal pocket.
All molecular surfaces were generated using GRASP [Nicholls, 1991].
The present invention will now be described only by way of example; in which
reference
will be made to the following Tables:
Table 1 shows the data collection, structure determination and refinement
statistics
Table 2 shows the structural coordinates of a polo domain.
In Table 2, from the left, the second column identifies the atom number; the
third
identifies the atom type; the fourth identifies the amino acid type; the sixth
identifies the residue
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number; the seventh identifies the x coordinates; the eighth identifies the y
coordinates; the ninth
identifies the z coordinates; the tenth identifies the occupancy; and the
eleventh identifies the
temperature factor.
s DETAILED DESCRIPTION OF THE INVENTION
GLOSSARY
Unless otherwise indicated, all terms used herein have the same meaning as
they would
to one skilled in the art of the present invention. Practitioners are
particularly directed to Current
Protocols in Molecular Biology (Ansubel) for definitions and terms of the art:
Polo Family Kinase
The polo family kinases are a family of cell cycle regulators that have been
shown to be
important for progression through the cell cycle (Lane, H. A., Trends in Cell
Biol. 1997, 7:63-
68). The family contains the following related but distinct members:
(1) Plk (human polo-like kinase) and its homologs Polo (Drosophila), cdc5 (S.
cerevisiae), Plxp (Xenopus), and Plot (S. pombe), (see GenBank sequences in
Accession Nos.
P53350 (human Plk), P52304 (Drosophila PoLo), P32562 (S. cerevisiae cdc5),
AAC60017 (Plxp
Xenopus), P50528 (S. pombe Plop);
(2) Prk (polo-related kinase; human) and its murine homolog Fnk (see GenBank
sequences in Accession Nos AAC50637 and AAC52191);
(3) Snk (serum-inducible kinase; murine) (see GenBank sequence in Accession
No.
P53351); and,
(4) Sak (serine threonine kinase) (see GenBank sequences in Accession Nos.
CAA73575
(human), AAC37648 (murine), and AAD19607 (Drosophila).
The polo family kinases are characterized by a kinase domain and one or two
conserved
sequences in the noncatalytic C-terminal domain i.e: the polo domain.
A polo family kinase may be derivable from a variety of sources, including
viruses,
bacteria, fungi, plants and animals. In a preferred embodiment a polo family
kinase is derivable
from a mammal. For example, a polo family kinase may be a human Sak polo
family kinase
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A polo family kinase in the present invention may be a wild type enzyme, or
part thereof,
or a mutant, variant or homolog, or part of such an enzyme.
The term "wild type" refers to a polypeptide having a primary amino acid
sequence that
is identical with the native enzyme (for example, the human enzyme).
The term "mutant" refers to a polypeptide having a primary amino acid sequence
which
differs from the wild type sequence by one or more amino acid additions,
substitutions or
deletions. Preferably, the mutant has at least 90% sequence identity with the
wild type sequence.
Preferably, the mutant has 20 mutations or less over the whole wild-type
sequence. More
preferably the mutant has 10 mutations or less, most preferably 5 mutations or
less over the
1o whole wild-type sequence.
The term "variant" refers to a naturally occurnng polypeptide that differs
from a wild-
type sequence. A variant may be found within the same species (i.e. if there
is more than one
isoform of the enzyme) or may be found within a different species. Preferably
the variant has at
least 90% sequence identity with the wild type sequence. Preferably, the
variant has 20
mutations or less over the whole wild-type sequence. More preferably, the
variant has 10
mutations or less, most preferably 5 mutations or less over the whole wild-
type sequence.
The term "part" indicates that the polypeptide comprises a fraction of the
wild-type
amino acid sequence. It may comprise one or more large contiguous sections of
sequence or a
plurality of small sections. The "part" may comprise a binding pocket as
described herein. The
polypeptide may also comprise other elements of sequence, for example, it may
be a fusion
protein with another protein (such as one which aids isolation or
crystallisation of the
polypeptide). Preferably the polypeptide comprises at least 50%, more
preferably at least 65%,
most preferably at least 80% of the wild-type sequence.
The term "homolog" means a polypeptide having a degree of homology with the
wild-
type amino acid sequence. The term "homology" refers to a degree of
complementarity. There
may be partial homology or complete homology. A sequence that is
"substantially homologous"
refers to a partially complementary sequence that at least partially inhibits
an identical sequence
from hybridizing to a target nucleic acid. Inhibition of hybridization of a
completely
complementary sequence to the target sequence may be examined using a
hybridization assay
3o (e.g. Southern or northern blot, solution hybridization, etc.) under
conditions of reduced
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1
stringency. A sequence that is substantially homologous or a hybridization
probe will compete
for and inhibit the binding of a completely homologous sequence to the target
sequence under
conditions of reduced stringency. However, conditions of reduced stringency
can be such that
non-specific binding is permitted, as reduced stringency conditions require
that the binding of
two sequences to one another be a specific (i.e., a selective) interaction.
The absence of non-
specific binding may be tested using a second target sequence which lacks even
a partial degree
of complementarity (e.g., less than about 30% homology or identity). The
substantially
homologous sequence or probe will not hybridize to the second non-
complementary target
sequence in the absence of non-specific binding.
1o The phrase "percent identity" or "% identity" refers to the percentage of
sequence
similarity found in a comparison of two or more amino acid sequences. Percent
identity can be
determined electronically using conventional programs, e.g., by using the
MEGALIGN program
(LASERGENE software package, DNASTAR). The MEGALIGN program can create
alignments between two or more amino acid sequences according to different
methods, e.g., the
Clustal Method. (Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) Gaps
of low or of
no homology between the two amino acid sequences are not included in
determining percentage
similarity.
In the present context, a homologous sequence is taken to include an amino
acid
sequence which may have at least 75, 85 or 90% identity, preferably at least
95 or 98% identity
2o to the wild-type sequence. The homologs will comprise the same sites (for
example, binding
pockets) as the subject amino acid sequence.
A sequence may have deletions, insertions or substitutions of amino acid
residues which
produce a silent change and result in a functionally equivalent enzyme.
Deliberate amino acid
substitutions may be made on the basis of similarity in polarity, charge,
solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues
as long as the
secondary binding activity of the substance is retained. For example,
negatively charged amino
acids include aspartic acid and glutamic acid; positively charged amino acids
include lysine and
arginine; and amino acids with uncharged polar head groups having similar
hydrophilicity values
include leucine, isoleucine, valine, glycine, alanine; asparagine, glutamine,
serine, threonine,
phenylalanine, and tyrosine.
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The polypeptide may also have a homologous substitution (substitution and
replacement
are both used herein to mean the interchange of an existing amino acid
residue, with an
alternative residue) i.e. like-for-like substitution such as basic for basic,
acidic for acidic, polar
for polar etc. Non-homologous substitution may also occur i.e. frorn one class
of residue to
another or alternatively involving the inclusion of unnatural amino acids such
as ornithine
(hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter
referred to as B),
norleucine ornithine (hereinafter referred to as O), pyriylalanine,
thienylalanine, naphthylalanine
and phenylglycine.
Polo Domain
1o A "polo domain" is a highly conserved sequence in the non-catalytic domain
of polo
family kinases. Figure 1 shows the sequences of polo domains from various polo
family kinases.
In the present invention the polo domain may be a polo domain of Plk, Polo,
cdc5, Plx,
Plo, Prk, Fnk, Snk, or Sak., preferably Sak.
Binding Pocket
IS "Binding pocket" refers to a region or site of a polo domain or molecular
complex thereof
that as a result of its shape, favorably associates with another region of the
polo domain or polo
family kinase or with a ligand or a part thereof. For example, it may comprise
a region
responsible for binding a ligand.
A "ligand" refers to a compound or entity that associates with a polo domain
or binding
2o pocket thereof including substrates or analogues or parts thereof,
effectors, or modulators of polo
family kinases, including inhibitors. A ligand may be designed rationally by
using a model
according to the present invention. Fox example, a ligand for Plk may be Golgi
Reassembly
Stacking Protein of 65kDa (GRASP65) (Lin Cy et al, Proc. Natl. Acad, Sci USA
2000, 7;
97(23): 12589-94), an alpha or beta tubulin (Feng, Y et al, Biochem J 1999
15;339 (Pt2): 435-
25 42); human cytomegalovirus (HCMV) pp65 lower matrix protein (Gallina, A: et
al J. Virol. 1999
73(2): 1468-78); and associated with peptidyl-prolyl isomerase (Pinl). A
ligand for Prk/Fnk and
Snk may be Cib, a Ca2+ and integrin-binding protein.
The term "binding pocket" (BP) also includes a homolog of the binding pocket
or a
portion thereof. As used herein, the term "homolog" in reference to a binding
pocket refers to a
3o binding pocket or a portion thereof which may have deletions, insertions or
substitutions of
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amino acid residues as long as the binding specificity is retained. In this
regard, deliberate amino
acid substitutions may be made on the basis of similarity in polarity; charge,
solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues
as long as the
binding specificity of the binding pocket is retained.
As used herein, the term "portion thereof" means the structural coordinates
corresponding
to a sufficient number of amino acid residues of a binding pocket (or homologs
thereof) that are
capable of associating with a ligand. For example, the structural coordinates
provided in a crystal
structure may contain a subset of the amino acid residues in a binding pocket
which may be
useful in the modelling and design of compounds that bind to the binding
pocket.
CRYSTAL
The invention provides crystal structures. As used herein, the term "crystal"
or
"crystalline" means a structure (such as a three dimensional (3D) solid
aggregate) in which the
plane faces intersect at definite angles and in which there is a regular
structure (such as internal
structure) of the constituent chemical species. Thus, the term "crystal" can
include any one of: a
solid physical crystal form such as an experimentally prepared crystal, a
crystal structure
derivable from the crystal (including secondary and/or tertiary and/or
quaternary structural
elements), a 2D andlor 3D model based on the crystal structure, a
representation thereof such as a
schematic representation thereof or a diagrammatic representation thereof, or
a data set thereof for
2o a computer. In one aspect, the crystal is usable in X-ray crystallography
techniques. Here, the
crystals used can withstand exposure to X-ray beams used to produce a
diffraction pattern data
necessary to solve the X-ray crystallographic structure. A crystal of a polo
domain or binding
pocket may be characterized as being capable of diffracting x-rays in a
pattern defined by one of
the crystal forms depicted in Blundel et al 1976, Protein Crystallography,
Academic Press.
The invention contemplates a crystal comprising a polo domain or binding
pocket thereof
of the invention.
In an embodiment, the invention relates to a crystal that is characterized as
follows:
(a) dimeric in nature;
(b) comprising a two-sheet, strand-exchange (3-fold.
3o The crystal may be further characterized by one or more of the following
properties:
CA 02373918 2002-02-28
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(a) a monomer comprising at its amino terminus five ~3-strands ((3i-X35, one a-
helix (aA),_and
a C-terminal (3-strand ((36);
(b) ~i-strands 6, 1, 2, and 3 from one monomer form a contiguous anti parallel
sheet with (3-
strands 4 and 5 from the second monomer;
(c) two (3-sheets pack with a crossing angle of 110°, orienting
hydrophobic surfaces inwards
and hydrophilic surfaces outwards;
(d) helix ocA, which is collinear with (3-strand 6 of the same monomer, buring
a large portion
of the non-overlapping hydrophobic (3-sheet surfaces;
(e) interactions involving helices ocA comprise a majority of the hydrophobic
core structure
1o and also the dimer interface;
0
(f) a total surface area buried by dimer formation is 2447 A2;
(g) the dimeric structure is clam like (60~ x 44~ x 2010, hinged at one end
through the
seamless association of (3-strands 3 from each monomer;
(h) a deep cavity of approximate dimensions 17A x 8.51 x 11.3A extending
inwards from
the mouth of the structure;
(i) an intermolecular salt interaction between Asp 868 and Lys 906; and
(j) an entranceway to the cavity that is relatively small (about 17th x 7.5t~)
and partitioned in
two by the contact of the Trp 853 side chains from each polypeptide of the
dimer.
A crystal of the invention may comprise amino acids residues Asp 868 and Lys
906.
2o Preferably the atoms of the Asp 868 and Lys 906 amino acid residues have
the structural
coordinates as set out in Table 2.
In an embodiment, a crystal of a polo domain of the invention belongs to space
group
P3212. The term "space group" refers to the lattice and symmetry of the
crystal. In a space group
designation the capital letter indicates the latdee type and the other symbols
represent symmetry
operations that can be carried out on the contents of the asymmetric unit
without changing its
appearance
A crystal of the invention may comprise a unit cell having the following unit
dimensions:
a = b = 51.78 (~0.05) A, c = 146.94 (~0.05) A. The term "unit cell" refers to
the smallest and
simplest volume element (i.e. parallelpiped-shaped block) of a crystal that is
completely
representative of the unit of pattern of the crystal. The unit cell axial
lengths are represented by a,
CA 02373918 2002-02-28
14
b, and cThose of skill in the art understand that a set of atomic coordinates
determined by X-ray
crystallography is not without standard error.
In a preferred embodiment, a crystal of the invention has the structural
coordinates as
shown in Table 2. As used herein, the term "structural coordinates" refers to
a set of values that
define the position of one or more amino acid residues with reference to a
system of axes. The
term refers to a data set that defines the three dimensional structure of a
molecule or molecules
(e.g. Cartesian coordinates, temperature factors, and occupancies). Structural
coordinates can be
slightly modified and still render nearly identical three dimensional
structures. A measure of a
unique set of structural coordinates is the root-mean-square deviation of the
resulting structure.
Structural coordinates that render three dimensional structures (in particular
a three dimensional
structure of a ligand binding pocket) that deviate from one another by a root-
mean-square
deviation of less than 5 A, 4 A, 3 A, 2 A, or 1.5 A may be viewed by a person
of ordinary skill in
the art as very similar.
Variations in structural coordinates may be generated because of mathematical
manipulations of the structural coordinates of a polo domain described herein.
For example, the
structural coordinates of Table 2 may be manipulated by crystallographic
permutations of the
structural coordinates, fractionalization of the structural coordinates,
integer additions or
substractions to sets of the structural coordinates, inversion of the
structural coordinates or any
combination of the above.
Variations in the crystal structure due to mutations, additions,
substitutions, and/or
deletions of the amino acids, or other changes in any of the components that
make up the crystal
may also account for modifications in structural coordinates. If such
modifications are within an
acceptable standard error as compared to the original structural coordinates,
the resulting
structure may be the same. Therefore, a ligand that bound to a polo domain or
binding pocket
thereof, would also be expected to bind to another polo domain or binding
pocket whose
structural coordinates defined a shape that fell within the acceptable error.
Such modified
structures of a polo domain or binding pocket thereof are also within the
scope of the invention.
Various computational analyses may be used to determine whether a molecule or
the
binding pocket thereof is sufficiently similar to all or parts of a polo
domain or binding pocket
CA 02373918 2002-02-28
thereof. Such analyses may be carried out using conventional software
applications and methods
as described herein.
A crystal of the invention may also be specifically characterised by the
parameters,
diffraction statistics andlor refinement statistics set out in Table 1.
5 With reference to a crystal of the present invention, residues in a binding
pocket may be
defined by their spatial proximity to a ligand in the crystal structure. For
example, a binding
pocket may be defined by their proximity to a modulator.
A crystal or secondary or three-dimensional structure of a polo domain or
binding pocket
thereof may be more specifically defined by one or more of the atomic contacts
of atomic
1o interactions in the crystal (e.g. between Asp 868 and Lys 9Q6). An atomic
interaction can be
defined by an atomic contact (more preferably, a specific atom of an amino
acid residue where
indicated) on the polo domain, and an atomic contact (more preferably, a
specific atom of an
amino acid residue where indicated) on the polo domain or ligand.
Illustrations of particular crystals of the invention are shown in Figures 3,
4, and 5.
15 A crystal of the invention includes a polo domain or binding pocket thereof
in association
with one or more moieties, including heavy-metal atoms i.e. a derivative
crystal, or one or more
ligands or molecules i.e. a co-crystal.
The term "associate", "association" or "associating" refers to a condition of
proximity
between a moiety (i.e. chemical entity or compound or portions or fragments
thereof), and a polo
2o domain or binding pocket thereof. The association may be non-covalent i.e.
where the
juxtaposition is energetically favored by for example, hydrogen-bonding, van
der Waals, or
electrostatic or hydrophobic interactions, or it may be covalent.
The term "heavy-metal atoms" refers to an atom that can be used to solve an x-
ray
crystallography phase problem, including but not limited to a transition
element, a lanthanide
metal, or an actinide metal: Lanthanide metals include elements with atomic
numbers between
57 and 71, inclusive. Actinide metals include elements with atomic numbers
between 89 and
103, inclusive.
Multiwavelength anomalous diffraction (MAD) phasing may be used to solve
protein
structures using selenomethionyl (SeMet) proteins. Therefore, a complex of the
invention may
CA 02373918 2002-02-28
16
comprise a crystalline polo domain or binding pocket with selenium on the
methionine residues
of the protein.
A crystal may comprise a complex between a polo domain or binding pocket
thereof and
one or more ligands or molecules. In other words the polo domain or binding
pocket may be
associated with one or more ligands or molecules in the crystal. The ligand
may be any
compound that is capable of stably and specifically associating with the polo
domain or binding
pocket. A ligand may, for example, be a modulator of a polo family kinase.
In an embodiment of the invention, a binding pocket is in association with a
cofactor in
the crystal. A "cofactor" refers to a molecule required for enzyme activity
and/or stability. For
example, the cofactor may be a metal ion.
Therefore, the present invention also provides:
(a) a crystal comprising a polo domain or binding pocket thereof and a
substrate or
analogue thereof;
(b) a crystal comprising a polo domain or binding pocket thereof and a ligand.
A structure of a complex of the invention may be defined by selected
intermolecular
contacts.
A crystal of the invention may enable the determination of structural data for
a ligand. In
order to be able to derive structural data for a ligand, it is necessary for
the molecule to have
sufficiently strong electron density to enable a model of the molecule to be
built using standard
techniques. For example, there should be sufficient electron density to allow
a model to be built
using XTALVIEW (McRee 1992 J. Mol. Graphics. 10 44-46).
METHOD OF MAKING A CRYSTAL
The present invention also provides a method of making a crystal according to
the
invention. The crystal may be formed from an aqueous solution comprising a
purified
polypeptide comprising a polo domain, in particular a polo family kinase or
part or fragment
thereof (e.g. a binding pocket). A method may utilize a purified polypeptide
comprising a
binding pocket to form a crystal. For example, amino acid residues 839 to 925
of Sak-a may be
used to prepare a polo domain structure of the invention.
CA 02373918 2002-02-28
17
The term "purified" in reference to a polypeptide, does not require absolute
purity such as
a homogenous preparation rather it represents an indication that the
polypeptide is relatively
purer than in the natural environment. Generally, a purified polypeptide is
substantially free of
other proteins, lipids, carbohydrates, or other materials with which it is
naturally associated,
preferably at a functionally significant level for example at least 85% pure;
more preferably at
least 95% pure, most preferably at least 99% pure. A skilled artisan can
purify a polypeptide
comprising using standard techniques fox protein purification. A substantially
pure polypeptide
will yield a single major band on a non-reducing polyacrylamide gel. Purity of
the polypeptide
can also be determined by amino-terminal amino acid sequence analysis.
l0 A polypeptide used in the method may be chemically synthesized in whole or
in part
using techniques that are well-known in the art. Alternatively, methods are
well known to the
skilled artisan to construct expression vectors containing a native or mutated
polo family kinase
coding sequence and appropriate transcriptional/translational control signals.
These methods
include in vitro recombinant DNA techniques, synthetic techniques, and ih vivo
recombination/genetic recombination. See for example the techniques described
in Sambrook et
al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor
Laboratory
press (1989)), and other laboratory textbooks. (See also Sarker et al,
Glycoconjugate J. 7:380,
1990; Sarker et al, Proc. Natl. Acad; Sci. USA 88:234-238, 1991, Sarker et al,
Glycoconjugate J.
11: 204-209, 1994; Hull et al, Biochem Biophys Res Commun 176:608, 1991 and
Pownall et al,
Genomics 12:699-704, 1992).
Crystals may be grown from an aqueous solution containing the purified
polypeptide by a
variety of conventional processes. These processes include batch, liquid,
bridge, dialysis, vapor
diffusion, and hanging drop methods. (See for example; McPherson, 1982 John
Wiley, New
York; McPherson, 1990, Eur. J. Biochem. 189: 1-23; Webber. 1991, Adv. Protein
Chem. 41:1-
36). Generally, native crystals of the invention are grown by adding
precipitants to the
concentrated solution of the polypeptide. The precipitants are added at a
concentration just below
that necessary to precipitate the protein. Water is removed by controlled
evaporation to produce
precipitating conditions, which are maintained until crystal growth ceases.
Derivative crystals of the invention can be obtained by soaking native
crystals in a
solution containing salts of heavy metal atoms. A complex of the invention can
be obtained by
CA 02373918 2002-02-28
1g
soaking a native crystal in a solution containing a compound that binds the
polypeptide, or they
can be obtained by co-crystallizing the polypeptide in the presence of one or
more compounds.
In order to obtain co-crystals with a compound which binds deep within the
tertiary structure of
the polypeptide it is necessary to use the second method.
Once the crystal is grown it can be placed in a glass capillary tube and
mounted onto a
holding device connected to an X-ray generator and an X-ray detection device.
Collection of X-
ray diffraction patterns are well documented by those skilled in the art (See
for example, Ducruix
and Geige, 1992, IRL Press, Oxford, England): A beam of X-rays enter the
crystal and diffract
from the crystal. An X-ray detection device can be utilized to record the
diffraction patterns
1o emanating from the crystal. Suitable devices include the Marr 345 imaging
plate detector system
with an RU200 rotating anode generator.
Multiwavelength anomalous diffraction (MAD) phasing using selenomethionyl
(SeMet)
proteins may be used to determine a crystal of the invention. Thus, the
invention contemplates a
method for determining a crystal structure of the invention using a
selenomethionyl derivative of
a polo domain or a binding pocket thereof.
Methods for obtaining the three dimensional structure of the crystalline form
of a
molecule or complex are described herein and known to those skilled in the art
(see Ducruix and
Geige 1992; IRL Press, Oxford, England). Generally, the x-ray crystal
structure is given by the
diffraction patterns. Each diffraction pattern reflection is characterized as
a vector and the data
2o collected at this stage determines the amplitude of each vector. The phases
of the vectors may be
determined by the isomorphous replacement method where heavy atoms soaked into
the crystal
are used as reference points in the X-ray analysis (see for example,
Otwinowski, 1991,
Daresbury, United Kingdom, 80-86). The phases of the vectors may also be
determined by
molecular replacement (see for example, Naraza, 1994, Proteins 11:281-296).
The amplitudes
and phases of vectors from the crystalline form are determined in accordance
with these methods
can be used to analyze other related crystalline polypeptides.
The unit cell dimensions and symmetry, and vector amplitude and phase
information can
be used in a Fourier transform function to calculate the electron density in
the unit cell i.e. to
generate an experimental electron density map. This may be accomplished using
the PHASES
3o package (Furey; 1990). Amino acid sequence structures are fit to the
experimental electron
CA 02373918 2002-02-28
19
density map (i.e. model building) using computer programs (e.g. Jones, TA. et
al, Acta
Crystallogr A47, 100-119, 1991). This structure can also be used to calculate
a theoretical
electron density map. The theoretical and experimental electron density maps
can be compared
and the agreement between the maps can be described by a parameter referred to
as R-factor. A
high degree of overlap in the maps is represented by a low value R-factor. The
R-factor can be
minimized by using computer programs that refine the structure to achieve
agreement between
the theoretical and observed electron density map. For example, the XPLOR
program, developed
by Brunger (1992, Nature 355:472-475) can be used for model refinement.
A three dimensional structure of the molecule or complex may be described by
atoms that
fit the theoretical electron density characterized by a minimum R value. Files
can be created for
the structure that defines each atom by coordinates in three dimensions.
MODEL
A crystal structure of the present invention may be used to make a model of a
polo
domain or binding pocket thereof. A model may, for example, be a structural
model or a
computer model. A model may represent the secondary, tertiary and/or
quaternary structure of
the binding pocket. The model itself may be in two or three dimensions. It is
possible for a
computer model to be in three dimensions despite the constraints imposed by a
conventional
computer screen, if it is possible to scroll along at least a pair of axes,
causing "rotation" of the
image.
As used herein, the term "modelling" includes the quantitative and qualitative
analysis of
molecular structure and/or function based on atomic structural information and
interaction
models. The term "modelling" includes conventional numeric-based molecular
dynamic and
energy minimization models, interactive computer graphic models, modified
molecular
mechanics models, distance geometry and other structure-based constraint
models.
Preferably, modelling is performed using a computer and may be further
optimized using
known methods, This is called modelling optimisation.
An integral step to an approach of the invention for designing modulators of a
subject
polo domain involves construction of computer graphics models of the domain
which can be
3o used to design pharmacophores by rational drug design. For instance, for a
modulator to interact
CA 02373918 2002-02-28
optimally with the subject domain, it will generally be desirable that it have
a shape which is at
least partly complimentary to that of a particular binding pocket of the
domain, as for example
those portions of the domain which are involved in recognition of a ligand.
Additionally, other
factors, including electrostatic interactions, hydrogen bonding, hydrophobic
interactions,
5 desolvation effects, and cooperative motions of ligand and domain, all
influence the binding
effect and should be taken into account in attempts to design bioactive
modulators.
As described herein, a computer-generated molecular model of the subject polo
domain
can be created. In preferred embodiments, at least the Ca,-carbon positions of
the polo domain
sequence of interest are mapped to a particular coordinate pattern, such as
the coordinates for a
1o polo domain shown in Table 2, by homology modeling, and the structure of
the protein and
velocities of each atom are calculated at a simulation temperature (To) at
which the docking
simulation is to be determined. Typically, such a protocol involves primarily
the prediction of
side-chain conformations in the modeled domain, while assuming a main-chain
trace taken from
a tertiary structure such as provided in Table 2 and the Figures. Computer
programs for
is performing energy minimization routines are commonly used to generate
molecular models. For
example, both the CHARMM (Brooks et al. (1983) J Comput Chem 4:187-217) and
AMBER
(Weiner et al (1981) J. Comput. Chem. 106: 765) algorithms handle all of the
molecular system
setup, force field calculation, and analysis (see also, Eisenfield et al.
(1991) Am J Physiol
261:C376-386; Lybrand (1991) J Pharm Belg 46:49-54; Froimowitz (1990)
Biotechniques
20 8:640-644; Burbam et al. (1990) Proteins 7:99-111; Pedersen (1985) Environ
Health Perspect
61:185-190; and Kini et al. (1991) J Biomol Struct Dyn 9:475-488): At the
heart of these
programs is a set of subroutines that; given the position of every atom in the
model, calculate the
total potential energy of the system and the force on each atom. These
programs may utilize a
starting set of atomic coordinates, such as the coordinates provided in Table
2, the parameters for
the various terms of the potential energy function, and a description of the
molecular topology
(the covalent structure). Common features of such molecular modeling methods
include:
provisions for handling hydrogen bonds and other constraint forces; the use of
periodic boundary
conditions; and provisions for occasionally adjusting positions; velocities,
or other parameters in
order to maintain or change temperature, pressure, volume, forces of
constraint, or other
3o externally controlled conditions.
CA 02373918 2002-02-28
21
Most conventional energy minimization methods use the input data described
above and
the fact that the potential energy function is an explicit, differentiable
function of Cartesian
coordinates, to calculate the potential energy and its gradient (which gives
the force on each
atom) for any set of atomic positions. This information can be used to
generate a new set of
coordinates in an effort to reduce the total potential energy and, by
repeating this process over
and over, to optimize the molecular structure under a given set of external
conditions. These
energy minimization methods are routinely applied to molecules similar to the
subject polo
domain.
In general, energy minimization methods can be carried out for a given
temperature, Ti,
l0 which may be different than the docking simulation temperature, To. Upon
energy minimization
of the molecule at Ti, coordinates and velocities of all the atoms in the
system are computed.
Additionally, the normal modes of the system are calculated. It will be
appreciated by those
skilled in the art that each normal mode is a collective, periodic motion,
with all parts of the
system moving in phase with each other, and that the motion of the molecule is
the superposition
of all normal modes. For a given temperature, the mean square amplitude of
motion in a
particular mode is inversely proportional to the effective force constant for
that mode, so that the
motion of the molecule will often be dominated by the low frequency
vibrations.
After the molecular model has been energy minimized at Ti, the system is
"heated" or
"cooled" to the simulation temperature, To, by carrying out an equilibration
run where the
2o velocities of the atoms are scaled in a step-wise manner until the desired
temperature, To, is
reached. The system is further equilibrated for a specified period of time
until certain properties
of the system, such as average kinetic energy, remain constant. The
coordinates and velocities of
each atom are then obtained from the equilibrated system.
Further energy minimization routines can also be earned out. For example, a
second
class of methods involves calculating approximate solutions to the constrained
EOM for the
protein. These methods use an iterative approach to solve for the Lagrange
multipliers and,
typically, only need a few iterations if the corrections required are small.
The most popular
method of this type, SHAKE (Ryckaert et al. (1977) J Comput Phys 23:327; and
Van Gunsteren
et al. (1977) Mol Phys 34:1311) is easy to implement and scales as O(N) as the
number of
3o constraints increases. Therefore, the method is applicable to molecules
such as the polo domains
CA 02373918 2002-02-28
22
of the present invention. An alternative method, RATTLE (Anderson (1983) J
Comput Phys
52:24) is based on the velocity version of the Verlet algorithm. Like SHAKE,
RATTLE is an
iterative algorithm and can be used to energy minimize the model of the
subject protein.
Overlays and super positioning with a three dimensional model of a polo domain
or
binding pocket thereof of the invention may be used for modelling
optimisation. Additionally
alignment and/or modelling can be used as a guide for the placement of
mutations on a polo
domain or binding pocket thereof to characterize the nature of the site in the
context of a cell.
The three dimensional structure of a new crystal may be modelled using
molecular
replacement. The term "molecular replacement" refers to a method that involves
generating a
to preliminary model of a molecule or complex whose structure coordinates are
unknown, by
orienting and positioning a molecule whose structure coordinates are known
within the unit cell
of the unknown crystal, so as best to account for the observed diffraction
pattern of the unknown
crystal. Phases can then be calculated from this model and combined with the
observed
amplitudes to give an approximate Fourier synthesis of the structure whose
coordinates are
unknown. This, in turn, can be subject to any of the several forms of
refinement to provide a
anal, accurate structure of the unknown crystal. Lattman, E., "Use of the
Rotation and
Translation Functions", in Methods in Enzymology, 115, pp. 55-77 (1985); M. G.
Rossmann,
ed., "The Molecular Replacement Method", Int. Sci. Rev. Ser.; No. 13, Gordon &
Breach, New
York, (1972).
2o Commonly used computer software packages for molecular replacement are X-
PLOR
(Brunger 1992, Nature 355: 472-475), AMORE (Navaza, 1994, Acta Crystallogr.
A50:157-163),
the CCP4 package (Collaborative Computational Project, Number 4, "The CCP4
Suite: Programs
for Protein Crystallography", Acta Cryst., Vol. D50, pp. 760-763, 1994), the
MERLOT package
(P.M.D. Fitzgerald, J. Appl. Cryst.; Vol. 21, pp. 273-278, 1988) and XTALVIEW
(McCree et al
(1992) J. Mol. Graphics 10: 44-46. It is preferable that the resulting
structure not exhibit a root-
a
mean-square deviation of more than 3 A.
Molecular replacement computer programs generally involve the following steps:
(1)
determining the number of molecules in the unit cell and defining the angles
between them (self
rotation function); (2) rotating the known structure against diffraction data
to define the
orientation of the molecules in the unit cell (rotation function); (3)
translating the known
CA 02373918 2002-02-28
23
structure in three dimensions to correctly position the molecules in the unit
cell (translation
function); (4) determining the phases of the X-ray diffraction data and
calculating an R-factor
calculated from the reference data set and from the new data wherein an R-
factor between 30-
50% indicates that the orientations of the atoms in the unit cell have been
reasonably determined
by the method; and (5) optionally, decreasing the R-factor to about 20% by
refining the new
electron density map using iterative refinement techniques known to those
skilled in the art
(refinement).
The quality of the model may be analysed using a program such as PRaCHECK or
3D-
Profiler [Laskowski et al 1993 J. Appl. Cryst. 26:283-291; Luthy R. et al,
Nature 356: 83-85,
l0 1992; and Bowie, J.U. et al, Science 253: 164-170, 1991]. Once any
irregularities have been
resolved, the entire structure may be further refined
Other molecular modelling techniques may also be employed in accordance with
this
invention. See, e.g., Cohen, N. C. et al, "Molecular Modelling Software and
Methods for
Medicinal Chemistry", J. Med. Chem., 33, pp. 883-894 (1990). See also, Navia,
M. A. and M. A.
Murcko, "The Use of Structural Information in Drug Design", Current Opinions
in Structural
Biology, 2, pp. 202-210 (1992).
Using the structural coordinates of crystals provided by the invention,
molecular
modelling may be used to determine the structural coordinates of a crystalline
mutant or
homolog of a polo domain or binding pocket thereof. By the same token a
crystal of the
invention can be used to provide a model of a ligand. Modelling techniques can
then be used to
approximate the three dimensional structure of ligand derivatives and other
components which
may be able to mimic the atomic contacts between a ligand and polo domain or
binding pocket.
COMPUTER FORMAT OF CRYSTALS/1VIODELS
Information derivable from a crystal of the present invention (for example the
structural
coordinates) and/or the model of the present invention may be provided in a
computer-readable
format.
Therefore, the invention provides a computer readable medium or a machine
readable
storage medium which comprises the structural coordinates of a polo domain or
binding pocket
CA 02373918 2002-02-28
24
thereof including all or any parts thereof, or Iigands including portions
thereof. Such storage
medium or storage medium encoded with these data are capable of displaying on
a computer
screen or similar viewing device, a three-dimensional graphical representation
of a molecule or
molecular complex which comprises such polo domain, binding pockets or
similarly shaped
homologous domains or binding pockets. Thus, the invention also provides
computerized
representations of the secondary or three-dimensional structures of a polo
domain or binding
pocket of the invention, including any electronic, magnetic, or
electromagnetic storage forms of
the data needed to define the structures such that the data will be computer
readable for purposes
of display and/or manipulation.
In an aspect the invention provides a computer for producing a three-
dimensional
representation of a molecule or molecular complex, wherein said molecule or
molecular complex
comprises a polo domain or binding pocket thereof defined by structural
coordinates of a polo
domain or binding pocket or structural coordinates of atoms of a ligand, or a
three-dimensional
representation of a homologue of said molecule or molecular complex, wherein
said homologue
comprises a polo domain, binding pocket or ligand that has a root mean square
deviation from
the backbone atoms not more than 1.5 angstroms wherein said computer
comprises:
(a) a machine-readable data storage medium comprising a data storage material
encoded
with machine readable data wherein said data comprises the structural
coordinates of a
polo domain or binding pocket thereof or a ligand according to Table 2;
(b) a working memory for storing instructions for processing said machine-
readable data;
(c) a central-processing unit coupled to said working memory and to said
machine-readable
data storage medium for processing said machine readable data into said three-
dimensional representation; and
(d) a display coupled to said central-processing unit for displaying said
three-dimensional
representation.
The invention also provides a computer for determining at least a portion of
the structural
coordinates corresponding to an X-ray diffraction pattern of a molecule or
molecular complex
wherein said computer comprises:
CA 02373918 2002-02-28
(a) a machine-readable data storage medium comprising a data storage material
encoded
with machine readable data wherein said data comprises the structure
coordinates
according to Table 2;
(b) a machine-readable data storage medium comprising a data storage material
encoded
5 with machine readable data wherein said data comprises an X-ray diffraction
pattern
of said molecule or molecular complex;
(c) a working memory for storing instructions for processing said machine-
readable data
of (a) and (b);
(d) a central-processing unit coupled to said working memory and to said
machine-
10 readable data storage medium of (a) and (b) for performing a Fourier
transform of the
machine readable data of (a) and for processing said machine readable data of
(b) into
structural coordinates; and
(e) a display coupled to said central-processing unit for displaying said
structural
coordinates of said molecule or molecular complex.
STRUCTURAL STUDIES
The present invention also provides a method for determining the secondary
and/or
tertiary structures of a polo domain or part thereof by using a crystal, or a
model according to the
2o present invention. The domain or part thereof may be any domain or part
thereof for which the
secondary and or tertiary structure is uncharacterised or incompletely
characterised. In a
preferred embodiment the domain shares (or is predicted to share) some
structural or functional
homology to a crystal of the present invention. For example; the domain may
show a degree of
structural homology over some or all parts of the primary amino acid sequence.
The polo domain may be a polo domain of a polo family kinase with a different
specificity for a ligand or substrate. Alternatively (or in addition) the
domain may be a polo
domain from a different species.
The domain may be from a mutant of a wild-type polo family kinase, in
particular Plk or
Sak. A mutant may arise naturally, or may be made artificially (for example
using molecular
biology techniques). The mutant may also not be "made" at all in the
conventional sense; but
CA 02373918 2002-02-28
26
merely tested theoretically using the model of the present invention. A mutant
may or may not be
functional.
Thus, using the model of the present invention, the effect of a particular
mutation on the
overall two and/or three dimensional structure of a polo domain and/or the
interaction between a
binding pocket of the enzyme and a ligand can be investigated.
Alternatively, the domain may perform an analogous function or be suspected to
show a
similar mechanism to a polo domain of a polo family kinase.
The domain may also be the same as the polo domain of the crystal, but in
association
with a different ligand (for example, modulator or inhibitor) or cofactor. In
this way it is possible
1o to investigate the effect of altering the ligand or compound with which the
polo domain is
associated on the structure of the binding pocket.
Secondary or tertiary structure may be determined by applying the structural
coordinates
of the crystal or model of the present invention to other data such as an
amino acid sequence, X-
ray crystallographic diffraction data, or nuclear magnetic resonance (NMR)
data. Homology
modeling, molecular replacement, and nuclear magnetic resonance methods using
these other
data sets are described below.
Homology modeling (also known as comparative modeling or knowledge-based
modeling) methods develop a three dimensional model from a sequence based on
the structures
of known proteins (i.e. a polo domain of the crystal of the invention). The
method utilizes a
computer model of the crystal of the present invention (the "known
structure"), a computer
representation of the amino acid sequence of the domain with an unknown
structure, and
standard computer representations of the structures of amino acids. The method
in particular
comprises the steps of; (a) identifying structurally conserved and variable
regions in the known
structure; (b) aligning the amino acid sequences of the known structure and
unknown structure
(c) generating co-ordinates of main chain atoms and side chain atoms in
structurally conserved
and variable regions of the unknown structure based on the coordinates of the
known structure
thereby obtaining a homology model; and (d) refining the homology model to
obtain a three
dimensional structure for the unknown structure. This method is well known to
those skilled in
the art (Greer, 1985, Science 228, 1055; Bundell et al 1988, Eur. J. Biochem.
172, 513; Knighton
et al., 1992, Science 258:130-135, http:l/biochem.vt.edu/courses/modeling/
homology.htn).
CA 02373918 2002-02-28
27
Computer programs that can be used in homology modelling are Quanta and the
Homology
module in the Insight II modelling package distributed by Molecular
Simulations Inc, or
MODELLER (Rockefeller University, www.iucr.ac.uk/sinris-top/logical/prg-
modeller.html).
In step (a) of the homology modelling method, a known structure is examined to
identify
the structurally conserved regions (SCRs) from which an average structure; or
framework, can be
constructed for these regions of the protein. Variable regions (VRs), in which
known structures
may differ in conformation; also must be identified. SCRs generally correspond
to the elements
of secondary structure, such as alpha-helices and beta-sheets, and to ligand-
and substrate-
binding sites (e:g. nucleotide binding sites). The VRs usually lie on the
surface of the proteins
i0 and form the loops where the main chain turns.
Many methods are available for sequence alignment of known structures and
unknown
structures. Sequence alignments generally are based on the dynamic programming
algorithm of
Needleman and Wunsch [J. Mol. Biol. 48: 442-453, 1970]. Current methods
include FASTA,
Smith-Waterman, and BLASTP, with the BLASTP method differing from the other
two in not
allowing gaps. Scoring of alignments typically involves construction of a
20x20 matrix in which
identical amino acids and those of similar character (i.e., conservative
substitutions) may be
scored higher than those of different character. Substitution chemes which
rnay be used to score
alignments include the scoring matrices PAM (Dayhoff et al., Meth. Enzymol.
91: 524-545,
1983), and BLOSUM (Henikoff and Henikoff; Proc. Nat. Acad. Sci. USA 89: 10915=
0919,
1992), and the matrices based on alignments derived from three-dimensional
structures including
that of Johnson and Overington (JO matrices) (J. Mol. Biol. 233: 716-738,
1993).
Alignment based solely on sequence may be used; however, other structural
features also
may be taken into account. In Quanta; multiple sequence alignment algorithms
are available that
may be used when aligning a sequence of the unknown with the known structures.
Four scoring
systems (i.e. sequence homology, secondary structure homology, residue
accessibility homology,
CA-CA distance homology) are available, each of which may be evaluated during
an alignment
so that relative statistical weights may be assigned.
When generating coordinates for the unknown structure, main chain atoms and
side chain
atoms, both in SCRs and VRs need to be modelled. A variety of approaches known
to those
skilled in the art may be used to assign co-ordinates to the unknown. In
particular, the co-
CA 02373918 2002-02-28
28
ordinates of the main chain atoms of SCRs will be transferred to the unknown
structure. VRs
correspond most often to the loops on the surface of the polypeptide and if a
loop in the known
structure is a good model for the unknown, then the main chain co-ordinates of
the known
structure may be copied. Side chain coordinates of SCRs and VRs are copied if
the residue type
in the unknown is identical to or very similar to that in the known structure.
For other side chain
coordinates, a side chain rotamer library may be used to define the side chain
coordinates. When
a good model for a loop cannot be found fragment databases may be searched for
loops in other
proteins that may provide a suitable model for the unknown. If desired, the
loop may then be
subjected to conformational searching to identify low energy conformers if
desired.
Once a homology model has been generated it is analyzed to determine its
correctness. A
computer program available to assist in this analysis is the Protein Health
module in Quanta
which provides a variety of tests. Other programs that provide structure
analysis along with
output include PROCHECK and 3D-Profiler [Luthy R. et al; Nature 356: 83-85;
1992; and
Bowie, J.U. et al, Science 253: 164-170, 1991]. Once any irregularities have
been resolved, the
entire structure may be further refined. Refinement may consist of energy
minimization with
restraints, especially for the SCRs. Restraints may be gradually removed for
subsequent
minimizations. Molecular dynamics may also be applied in conjunction with
energy
minimization.
Molecular replacement involves applying a known structure to solve the X-ray
2o crystallographic data set of a polypeptide of unknown structure. The method
can be used to
define the phases describing the X-ray diffraction data of a polypeptide of
unknown structure
when only the amplitudes are known. Thus in an embodiment of the invention, a
method is
provided for determining three dimensional structures of polypeptides with
unknown structure
by applying the structural coordinates of a crystal of the present invention
to provide an X-ray
crystallographic data set for a polypeptide of unknown structure, and (b)
determining a low
energy conformation of the resulting structure.
The structural coordinates of the crystal of the present invention may be
applied to
nuclear magnetic resonance (NMR) data to determine the three dimensional
structures of
polypeptides with uncharacterised or incompletely characterised sturcture.
(See for example,
Wuthrich, 1986, John Wiley and Sons; New York: 176-199; Pflugrath et al.,
1986, J. Molecular
CA 02373918 2002-02-28
29
Biology 189: 383-386; Kline et al., 1986 J. Molecular Biology 189:377-382).
While the
secondary structure of a polypeptide may often be determined by NMR data, the
spatial
connections between individual pieces of secondary structure are not as
readily determined. The
structural coordinates of a polypeptide defined by X-ray crystallography can
guide the NMR
spectroscopist to an understanding of the spatial interactions between
secondary structural
elements in a polypeptide of related structure. Information on spatial
interactions between
secondary structural elements can greatly simplify Nuclear Overhauser Effect
(NOE) data from
two-dimensional NMR experiments. In addition, applying the structural
coordinates after the
determination of secondary structure by NMR techniques simplifies the
assignment of NOE's
1o relating to particular amino acids in the polypeptide sequence and does not
greatly bias the NMR
analysis of polypeptide structure.
In an embodiment, the invention relates to a method of determining three
dimensional
structures of domains with unknown structures, by applying the structural
coordinates of a
crystal of the present invention to nuclear magnetic resonance (NMR) data of
the unknown
structure. This method comprises the steps of: (a) determining the secondary
structure of an
unknown structure using NMR data; and (b) simplifying the assignment of
through-space
interactions of amino acids. The term " through-space interactions" defines
the orientation of the
secondary structural elements in the three dimensional structure and the
distances between amino
acids from different portions of the amino acid sequence. The term
"assignment" defines a
2o method of analyzing NMR data and identifying which amino acids give rise to
signals in the
NMR spectrum.
SCREENING METHOD
Another aspect of the present invention concerns molecular models, in
particular three-
dimensional molecular models of polo domains, and their use as templates for
the design of
agents able to mimic or inhibit the activity of a polypeptide comprising a
polo domain.
In certain embodiments, the present invention provides a method of screening
for a ligand
that associates with a polo domain or binding pocket and/or modulates the
function of a polo
3o family kinase by using a crystal or a model according to the present
invention. The method may
CA 02373918 2002-02-28
involve investigating whether a test compound is capable of associating with
or binding a polo
domain or binding pocket thereof, and/or inhibiting or enhancing interactions
of atomic contacts
in a polo domain or binding pocket thereof.
In accordance with an aspect of the present invention, a method is provided
for screening
5 for a ligand capable of binding to a polo domain or a binding pocket
thereof, wherein the method
comprises using a crystal or model according to the invention.
In another aspect, the invention relates to a method of screening for a ligand
capable of
binding to a polo domain or binding pocket thereof, wherein the polo domain or
binding pocket
thereof is defined by the structural coordinates given herein, the method
comprising contacting
to the polo domain or binding pocket thereof with a test compound and
determining if the test
compound binds to the polo domain or binding pocket thereof.
In one embodiment, the present invention provides a method of screening for a
test
compound capable of interacting with one or more key amino acid residues of a
binding pocket
of a polo domain.
is Another aspect of the invention provides a process comprising the steps of:
(a) performing a method of screening for a ligand described above;
(b) identifying one or more ligands capable of binding to a binding pocket;
and
(c) preparing a quantity of said one or more ligands.
A further aspect of the invention provides a process comprising the steps of;
20 (a) performing a method of screening for a ligand as described above;
(b) identifying one or more ligands capable of binding to a binding pocket;
and
(c) preparing a pharmaceutical composition comprising said one or more
ligands.
Once a test compound capable of interacting with one or more key amino acid
residues in
a binding pocket of a polo domain has been identified, further steps may be
carried out either to
25 select and/or modify compounds and/or to modify existing compounds, to
modulate the
interaction with the key amino acid residues in the binding pocket.
Yet another aspect of the invention provides a process comprising the steps
of;
(a) performing the method of screening for a ligand as described above;
(b) identifying one or more ligands capable of binding to a binding pocket;
30 (c) modifying said one or more ligands capable of binding to a binding
pocket;
CA 02373918 2002-02-28
31
(d) performing said method of screening for a ligand as described above; and
(e) optionally preparing a pharmaceutical composition comprising said one or
more
ligands
As used herein, the term "test compound" means any compound which is
potentially
capable of associating with a binding pocket, and/or inhibiting or enhancing
interactions of
atomic contacts in a binding pocket. If, after testing; it is determined that
the test compound does
bind to the binding pocket and/or inhibits or enhances interactions of atomic
contacts in a
binding contact, it is known as a "ligand".
The test compound may be designed or obtained from a library of compounds
which may
to comprise peptides, as well as other compounds, such as small organic
molecules and particularly
new lead compounds. By way of example, the test compound may be a natural
substance, a
biological macromolecule, or an extract made from biological materials such as
bacteria, fungi,
or animal (particularly mammalian) cells or tissues, an organic or an
inorganic molecule, a
synthetic test compound, a semi-synthetic test compound, a carbohydrate, a
monosaccharide, an
oligosaccharide or polysaccharide, a glycolipid, a glycopeptide, a saponin, a
heterocyclic
compound, a structural or functional mimetic, a peptide, a peptidomimetic, a
derivatised test
compound, a peptide cleaved from a whole protein, or a peptides synthesised
synthetically (such
as, by way of example, either using a peptide synthesizer or by recombinant
techniques or
combinations thereof), a recombinant test compound, a natural or a non-natural
test compound, a
2o fusion protein or equivalent thereof and mutants, derivatives or
combinations thereof.
The increasing availability of biomacromolecule structures of potential
pharmacophoric
molecules that have been solved crystallographically has prompted the
development of a variety
of direct computational methods for molecular design, in which the steric and
electronic
properties of substrate binding sites are use to guide the design of potential
ligands (Cohen et al.
(1990) J. Med. Cam. 33: 883-894; Kuntz et al. (1982) J. Mol. Biol 161: 269-
288; DesJarlais
(1988) J. Med. Cam. 31: 722-729; Bartlett et aT. (1989) (Spec. Publ., Roy.
Soc. Chem.) 78: 182-
196; Goodford et al. (1985) J. Med. Cam. 28: 849-857; DesJarlais et al. J.
Med. Cam. 29: 2149-
2153). Directed methods generally fall into two categories: (1) design by
analogy in which 3-D
structures of known molecules (such as from a crystallographic database) are
docked to the
3o domain structure and scored for goodness-of-fit; and (2) de novo design, in
which the ligand
CA 02373918 2002-02-28
32
model is constructed piece-wise in the domain structure. The latter approach,
in particular, can
facilitate the development of novel molecules; uniquely designed to bind to
the subject domain.
The test compound may be screened as part of a library or a data base of
molecules. Data
bases which may be used include ACD (Molecular Designs Limited), NCI (National
Cancer
Institute), CCDC (Cambridge Crystallographic Data Center), CAST (Chemical
Abstract
Service), Derwent (Derwent Information Limited), Maybridge (Maybridge Chemical
Company
Ltd), Aldrich (Aldrich Chemical Company), DOCK (University of California in
San Francisco),
and the Directory of Natural Products (Chapman & Hail). Computer programs such
as
CONCORD (Tripos Associates) or DB-Converter (Molecular Simulations Limited)
can be used
to convert a data set represented in two dimensions to one represented in
three dimensions.
Test compounds may tested for their capacity to fit spatially into a binding
pocket. As
used herein, the term "fits spatially" means that the three-dimensional
structure of the test
compound is accommodated geometrically in a cavity of a binding pocket. The
test compound
can then be considered to be a ligand.
A favourable geometric fit occurs when the surface area of the test compound
is in close
proximity with the surface area of the cavity of a binding pocket without
forming unfavorable
interactions. A favourable complementary interaction occurs where the test
compound interacts
by hydrophobic, aromatic, ionic, dipolar, or hydrogen donating and accepting
forces.
Unfavourable interactions may be steric hindrance between atoms in the test
compound and
atoms in the binding pocket.
If a model of the present invention is a computer model, the test compounds
may be
positioned in a binding pocket through computational docking. If; on the other
hand, the model
of the present invention is a structural model, the test compounds may be
positioned in the
binding pocket by, for example, manual docking.
As used herein the term "docking" refers to a process of placing a compound in
close
proximity with a binding packet, or a process of finding low energy
conformations of a test
compound/ binding pocket complex.
In an illustrative embodiment, the design of potential polo domain ligands
begins from
the general perspective of shape complimentary for an active site and
substrate specificity
3o subsites of the domain; and a search algorithm is employed which is capable
of scanning a
CA 02373918 2002-02-28
33
database of small molecules of known three-dimensional structure for
candidates which fit
geometrically into the target protein site. It is not expected that the
molecules found in the shape
search will necessarily be leads themselves, since no evaluation of chemical
interaction
necessarily be made during the initial search. Rather, it is anticipated that
such candidates might
act as the framework for further design, providing molecular skeletons to
which appropriate
atomic replacements can be made. Of course, the chemical complimentarity of
these molecules
can be evaluated, but it is expected that atom types will be changed to
maximize the electrostatic,
hydrogen bonding, and hydrophobic interactions with the protein. Most
algorithms of this type
provide a method for finding a wide assortment of chemical structures that are
complementary to
to the shape of a binding pocket of the subject domain. Each of a set of small
molecules from a
particular data-base, such as the Cambridge Crystallographic Data Bank (CCDB)
(Allen et al.
(1973) J. Chem. Doc. 13: 119), is individually docked to the binding pocket or
site of a polo
domain, in particular a Sak or Plk polo domain; in a number of geometrically
permissible
orientations with use of a docking algorithm. In a preferred embodiment; a set
of computer
algorithms called DOCK, can be used to characterize the shape of invaginations
and grooves that
form active sites and recognition surfaces of a subject structure (Kuntz et
al. (1982) J. Mol. Biol
161: 269-288). The program can also search a database of small molecules for
templates whose
shapes are complementary to particular binding pockets or sites of a structure
(DesJarlais et al.
(1988) J Med Chem 31: 722-729). These templates normally require modification
to achieve
good chemical and electrostatic interactions (DesJarlais et al. (1989) ACS
Symp Ser 413: 60-69).
However, the program has been shown to position accurately known cofactors for
ligands based
on shape constraints alone.
The orientations are evaluated for goodness-of-fit and the best are kept for
further
examination using molecular mechanics programs, such as AMBER or CHARMM. Such
algorithms have previously proven successful in finding a variety of molecules
that are
complementary in shape to a given binding site of a structure, and have been
shown to have
several attractive features. First, such algorithms can retrieve a remarkable
diversity of
molecular architectures. Second, the best structures have, in previous
applications to other
proteins, demonstrated impressive shape complementarity over an extended
surface area. Third,
CA 02373918 2002-02-28
34
the overall approach appears to be quite robust with respect to small
uncertainties in positioning
of the candidate atoms.
Goodford (1985, J Med Chem 28:849-857) and Boobbyer et al. (1989, J Med Chem
32:1083-1094) have produced a computer program (GRID) which seeks to determine
regions of
high affinity for different chemical groups (termed probes) on the molecular
surface of the
binding site. GRID hence provides a tool for suggesting modifications to known
ligands that
might enhance binding. It may be anticipated that some of the sites discerned
by GRID as
regions of high affinity correspond to "pharrnacophoric patterns" determined
inferentially from a
series of known ligands. As used herein, a pharmacophorie pattern is a
geometric arrangement of
l0 features of the anticipated ligand that is believed to be important for
binding. Attempts have
been made to use pharmacophoric patterns as a search screen for novel ligands
(lakes et al.
(1987) J Mol Graph 5:41-48; Brint et al. (1987) J Mol Graph 5:49-56; lakes et
al. (1986) J Mol
Graph 4:12-20); however, the constraint of steric and "chemical" fit in the
putative (and possibly
unknown) binding pocket or site is ignored. Goodsell and Olson (1990,
Proteins: Struct Funct
Genet 8:195-202) have used the Metropolis (simulated annealing) algorithm to
dock a single
knowm ligand into a target protein. They allow torsional flexibility in the
ligand and use GRID
interaction energy maps as rapid lookup tables for computing approximate
interaction energies.
Given the large number of degrees of freedom available to the ligand, the
Metropolis algorithm
is time-consuming and is unsuited to searching a candidate database of a few
thousand small
molecules.
Yet a further embodiment of the present invention utilizes a computer
algorithm such as
CLIX which searches such databases as CCDB for small molecules which can be
oriented in a
binding pocket or site in a way that is both sterically acceptable and has a
high likelihood of
achieving favorable chemical interactions between the candidate molecule and
the surrounding
amino acid residues. The method is based on characterizing a binding pocket in
terms of an
ensemble of favorable binding positions for different chemical groups and then
searching for
orientations of the candidate molecules that cause maximum spatial coincidence
of individual
candidate chemical groups with members of the ensemble: The current
availability of computer
power dictates that a computer-based search for novel ligands follows a
breadth-first strategy. A
3o breadth-first strategy aims to reduce progressively the size of the
potential candidate search
CA 02373918 2002-02-28
space by the application of increasingly stringent criteria, as opposed to a
depth-first strategy
wherein a maximally detailed analysis of one candidate is performed before
proceeding to the
next. CLIX conforms to this strategy in that its analysis of binding is
rudimentary -it seeks to
satisfy the necessary conditions of steric fit and of having individual groups
in "correct" places
5 for bonding, without imposing the sufficient condition that favorable
bonding interactions
actually occur. A ranked "shortlist" of molecules, in their favored
orientations, is produced
which can then be examined on a molecule-by-molecule basis, using computer
graphics and
more sophisticated molecular modeling techniques. CLIX is also capable of
suggesting changes
to the substituent chemical groups of the candidate molecules that might
enhance binding.
1o The algorithmic details of CLIX is described in Lawerence et al. (1992)
Proteihs 12:31-
41, and the CLIX algorithm can be summarized as follows. The GRID program is
used to
determine discrete favorable interaction positions (termed target sites) in
the binding pocket or
site of the protein for a wide variety of representative chemical groups. :For
each candidate
ligand in the CCDB an exhaustive attempt is made to make coincident, in a
spatial sense in the
15 binding site of the protein, a pair of the candidate's substituent chemical
groups with a pair of
corresponding favorable interaction sites proposed by GRll~. All possible
combinations of pairs
of ligand groups with pairs of GRID sites are considered during this
procedure. Upon locating
such coincidence, the program rotates the candidate ligand about the two pairs
of groups and
checks for steric hindrance and coincidence of other candidate atomic groups
with appropriate
20 target sites. Particular candidate/orientation combinations that are good
geometric fits in the
binding site and show sufficient coincidence of atomic groups with GRID sites
are retained.
Consistent with the breadth-first strategy, this approach involves simplifying
assumptions. Rigid protein and small molecule geometry is maintained
throughout. As a first
approximation rigid geometry is acceptable as the energy minimized coordinates
of a polo
25 domain, in particular a Sak polo domain deduced structure, as described
herein, describe an
energy minimum for the molecule, albeit a local one. If the surface residues
of the site of interest
are not involved in crystal contacts then the crystal configuration of those
residues is used merely
as a starting point for energy minimization, and potential solution structures
for those residues
determined. The deduced structure described herein should reasonably mimic the
mean solution
3o configuration.
CA 02373918 2002-02-28
36
A further assumption implicit in CLIX is that the potential ligand, when
introduced into
the binding pocket or site, does not induce change in the protein's
stereochemistry or partial
charge distribution and so alter the basis on which the GRIZ7 interaction
energy maps were
computed. It must also be stressed that the interaction sites predicted by
GRID are used in a
positional and type sense only, i.e., when a candidate atomic group is placed
at a site predicted as
favorable by GRID, no check is made to ensure that the bond geometry, the
state of protonation,
or the partial charge distribution favors a strong interaction between the
protein and that group.
Such detailed analysis should form part of more advanced modeling of
candidates identified in
the CLIX shortlist.
1o Yet another embodiment of a computer-assisted molecular design method for
identifying
ligands of a polo domain comprises the de novo synthesis of potential ligands
by algorithmic
connection of small molecular fragments that will exhibit the desired
structural and electrostatic
complementaxity with a polo domain or binding pocket thereof. The methodology
employs a
large template set of small molecules with are iteratively pieced together in
a model of a polo
domain or binding pocket. Each stage of ligand growth is evaluated according
to a molecular
mechanics-based energy function, which considers van der Waals and coulombic
interactions,
internal strain energy of the lengthening ligand, and desolvation of both
ligand and domain. The
search space can be managed by use of a data tree which is kept under control
by pruning
according to the binding criteria.
In an illustrative embodiment, the search space is limited to consider only
amino acids
and amino acid analogs as the molecular building blocks. Such a methodology
generally
employs a large template set of amino acid conformations, though need not be
restricted to just
the 20 natural amino acids, as it can easily be extended to include other
related fragments of
interest to the medicinal chemist, e.g. amino acid analogs. The putative
ligands that result from
this construction method are peptides and peptide-like compounds rather than
the small organic
molecules that are typically the goal of drug design research. The appeal of
the peptide building
approach is not that peptides are preferable to organics as potential
pharmaceutical agents, but
rather that: (1) they can be generated relatively rapidly de novo; (2) their
energetics can be
studied by well-parameterized force field methods; (3) they are much easier to
synthesize than
3o are most organics; and (4) they can be used in a variety of ways, for
peptidomimetic ligand
CA 02373918 2002-02-28
37
design, protein-protein binding studies, and even as shape templates in. the
more commonly used
3D organic database search approach described above.
Such a de novo peptide design method has been incorporated in a software
package called
GROW (Moon et al. (1991) Proteins 11:314-328). In a typical design session,
standard
interactive graphical modeling methods are employed to define the structural
environment in
which GROW is to operate. For instance, environment could be an active site
binding pocket of
a polo domain, in particular a Sak or Plk polo domain, or it could be a set of
features on the
protein's surface to which the user wishes to bind a peptide-like molecule.
The GROW program
then operates to generate a set of potential ligand molecules: Interactive
modeling methods then
1o come into play again, for examination of the resulting molecules, and for
selection of one or
more of them for further refinement.
To illustrate, GROW operates on an atomic coordinate file generated by the
user in the
interactive modeling session; such as the coordinates provided in Table 2 plus
a small fragment
{e.g., an acetyl group) positioned in the active site to provide a starting
point for peptide growth.
These are referred to as "site" atoms and "seed" atoms, respectively. A second
file provided by
the user contains a number of control parameters to guide the peptide growth
(Moon et al. (1991)
Proteins 11:314-328).
The operation of the GROW algorithm is conceptually fairly simple. GROW
proceeds in
an iterative fashion, to systematically attach to the seed fragment each amino
acid template in a
large preconstructed library of amino acid conformations. When a template has
been attached, it
is scored for goodness-of-fit to the polo domain or binding pocket thereof;
and then the next
template in the library is attached to the seed. After all the templates have
been tested, only the
highest scoring ones are retained for the next level of growth. This procedure
is repeated for the
second growth level; each library template is attached in turn to each of the
bonded seed/amino
acid molecules that were retained from the first step, and is then scored.
Again; only the best of
the bonded seed/dipeptide molecules that result are retained for the third
level of growth. The
growth of peptides can proceed in the N-to-C direction only, the reverse
direction only, or in
alternating directions, depending on the initial control specifications
supplied by the user.
Successive growth levels therefore generate peptides that are lengthened by
one residue. The
3o procedure terminates when the user-defined peptide length has been reached,
at which point the
CA 02373918 2002-02-28
38
user can select from the constructed peptides those to be studied further. The
resulting data
provided by the GROW procedure includes not only residue sequences and scores,
but also
atomic coordinates of the peptides, related directly to the coordinate system
of the domain site
atoms.
In yet another embodiment, potential pharmacophoric compounds can be
determined
using a method based on an energy minimization-quenched molecular dynamics
algorithm for
determining energetically favorable positions of functional groups in the
binding pockets of the
subject polo domain. The method can aid in the design of molecules that
incorporate such
functional groups by modification of known ligands or de novo construction.
1o For example, the multiple copy simultaneous search method (MOSS) described
by
Miranker et al. (1991) Proteins 11: 29-34. To determine and characterize a
local minima of a
functional group in the forcefield of the protein, multiple copies of selected
functional groups are
first distributed in a binding pocket of interest on the polo domain. Energy
minimization of these
copies by molecular mechanics or quenched dynamics yields the distinct local
minima. The
neighborhood of these minima can then be explored by a grid search or by
constrained
minimization. In one embodiment, the MCSS method uses the classical time
dependent Hartee
(TDH) approximation to simultaneously minimize or quench many identical groups
in the
forcefield of the protein.
Implementation of the MCSS algorithm requires a choice of functional groups
and a
2o molecular mechanics model for each of them. Groups must be simple enough to
be easily
characterized and manipulated (3-6 atoms, few or no dihedral degrees of
freedom), yet complex
enough to approximate the steric and electrostatic interactions that the
functional group would
have in binding to the pocket or site of interest in the polo domain: A
preferred set is, for
example, one in which most organic molecules can be described as a collection
of such groups
(Patai's Guide to the Chemistry of Functional Groups, ed. S. Patai (New York:
John Wiley, and
Sons, (1989)). This includes fragments such as acetonitrile, methanol,
acetate, methyl
ammonium, dimethyl ether, methane, and acetaldehyde.
Determination of the local energy minima in the binding pocket or site
requires that many
starting positions be sampled. This can be achieved by distributing, for
example, 1;000-5,000
3o groups at random inside a sphere centered on the binding site; only the
space not occupied by
CA 02373918 2002-02-28
39
the protein needs to be considered. If the interaction energy of a particular
group at a certain
location with the protein is more positive than a given cut-off (e.g. 5.O
kcal/mole) the group is
discarded from that site. Given the set of starting positions, all the
fragments are minimized
simultaneously by use of the TDH approximation (Elber et al. (1990) JAm Chem
Soc 112: 9161-
9175). In this method, the forces on each fragment consist of its internal
forces and those due to
the protein. The essential element of this method is that the interactions
between the fragments
are omitted and the forces on the protein are normalized to those due to a
single fragment. In this
way simultaneous minimization or dynamics of any' number of functional groups
in the field of a
single protein can be performed.
1o Minimization is performed successively on subsets of, for example 100, of
the randomly
placed groups. After a certain number of step intervals, such as 1,000
intervals, the results can
be examined to eliminate groups converging to the same minimum. This process
is repeated
until minimization is complete (e.g: RMS gradient of 0:01 kcallrnole/C). ;
Thus the resulting
energy minimized set of molecules comprises what amounts to a set of
disconnected fragments
in three dimensions representing potential pharrnacophores.
The next step then is to connect the pharmacophoric pieces with spacers
assembled from
small chemical entities (atoms, chains, or ring moieties). In a preferred
embodiment, each of the
disconnected can be linked in space to generate a single molecule using such
computer programs
as, for example; NEWLEAD (Tschinke et al. (1993) J Med Chem 36: 3863,3870).
The
2o procedure adopted by NEWLEAD executes the following sequence of commands
(1) connect
two isolated moieties, (2) retain the intermediate solutions for further
processing, (3) repeat the
above steps for each of the intermediate solutions until no disconnected units
are found, and (4)
output the final solutions, each of which is single molecule. Such a program
can use for
example, three types of spacers: library spacers, single-atom spacers, and
fuse-ring spacers. The
library spacers are optimized structures of small: molecules such as ethylene,
benzene and
methylamide. The output produced by programs such as NEWLEAD consist of a set
of
molecules containing the original fragments now connected by spacers. The
atoms belonging-to
the input fragments maintain their original orientations in space. The
molecules are chemically
plausible because of the simple makeup of the spacers and functional groups,
and energetically
acceptable because of the rejection of solutions with van-der Waals radii
violations.
CA 02373918 2002-02-28
A screening method of the present invention may comprise the following steps:
(i) generating a computer model of a binding pocket using a crystal according
to the
invention;
(ii) docking a computer representation of a test compound with the computer
model;
5 (iii) analysing the fit of the compound in the binding pocket.
In an aspect of the invention, a method is provided comprising the following
steps:
(a) docking a computer representation of a structure of a test compound into a
computer
representation of a binding pocket of a polo domain in accordance with the
invention
using a computer program, or by interactively moving the representation of the
test
1o compound into the representation of the binding pocket;
(b) characterizing the geometry and the complementary interactions formed
between the
atoms of the binding pocket and the compound; optionally
(c) searching libraries for molecular fragments which can fit into the empty
space
between the compound and the binding pocket and can be linked to the compound;
15 and
(d) linking the fragments found in (c) to the compound and evaluating the new
modified
compound.
In an embodiment of the invention, a method is provided which comprises the
following
steps:
20 (a) docking a computer representation of a test compound from a computer
data base
with a computer representation of a selected binding pocket on a polo domain
defined
in accordance with the invention to define a complex;
(b) determining a conformation of the complex with a favorable fit and
favourable
complementary interactions; and
25 (c) identifying test compounds that best fit the selected binding pocket as
potential
modulators of the polo domain.
The method may be applied to a plurality of test compounds, to identify those
that best fit
the selected site.
The model used in the screening method may comprise a binding pocket either
alone or
3o in association with one or more ligands and/or cofactors. For example, the
model may comprise
CA 02373918 2002-02-28
41
the binding pocket in association with a nucleotide (or analogue thereof), a
substrate (or
analogue thereof), andlor modulator.
If the model comprises an unassociated binding pocket, then the selected site
under
investigation may be the binding pocket itself. The test compound may, for
example, mimic a
known ligand (e.g. substrate) for a polo family kinase in order to interact
with the binding
pocket. The selected site may alternatively be another site on the polo domain
or polo family
kinase.
If the model comprises an associated binding pocket, for example a binding
pocket in
association with a ligand, the selected site may be the binding pocket ox a
site made up of the
to binding pocket and the complexed ligand, or a site on the ligand itself.
The test compound may
be investigated for its capacity to modulate the interaction with the
associated molecule.
A test compound (or plurality of test compounds) may be selected on the basis
of their
similarity to a known ligand for a polo domain, in particular a Sak or Plk
polo domain. For
example, the screening method may comprise the following steps:
(i) generating a computer model of a binding pocket in complex with a ligand;
(ii) searching for a test compound with a similar three dimensional structure
and/or
similar chemical groups; and
(iii) evaluating the fit of the test compound in the binding pocket.
Searching may be corned out using a database of computer representations of
potential
2o compounds, using methods known in the art.
The present invention also provides a method for designing a ligand for a polo
domain. It
is well known in the art to use a screening method as described above to
identify a test
compound with promising fit, but then to use this test compound as a starting
point to design a
ligand with improved fit to the model. Such techniques are known as "structure-
based ligand
design" (See Kuntz et al., 1994, Acc. Chem. Ices. 27:117; Guida, 1994; Current
Opinion in Struc.
Biol. 4: 777; and Colman; 1994, Current Opinion in' Struc. Biol. 4: 868, for
reviews of structure-
based drug design and identification;and Kuntz et al 1982, J. Mol. Biol.
162:269; Kuntz et al.,
1994, Acc. Chem. Res. 27: 117; lVleng et al., 1992, J. Compt. Chem. 13: 505;
Bohm, 1994, J.
Comp. Aided Molec. Design 8: 623 for methods of structure-based modulator
design).
CA 02373918 2002-02-28
42
Examples of computer programs that may be used for structure-based ligand
design are
CAVEAT (Bartlett et al" 1989; in "Chemical and Biological Problems in
Molecular
Recognition", Roberts, S.M. Ley; S.V.; Campbell, N.M. eds; Royal Society of
Chemistry:
Cambridge, pp 182-196); FLOG (Miller et al., 1994, J. Comp. Aided Molec.
Design 8:153);
PRO Modulator (Clark et al.; 1995 J. Comp. Aided Molec. Design 9:13); MOSS
(Miranker and
Karplus, 1991, Proteins: Structure, Fuction, and Genetics 8:195); and, GRID
(Goodford, 1985, J.
Med. Chem. 28:849).
The method may comprise the following steps:
(l) docking a model of a test compound with a model of a binding pocket;
o (ii) identifying one or more groups on the test compound which may be
modified to
improve their fit in the binding pocket;
(iii) replacing one or more identified groups to produce a modified test
compound
model; and
(iv) docking the modified test compound model with the model of the binding
pocket.
Evaluation of fit may comprise the following steps:
(a) mapping chemical features of a test compound such as by hydrogen bond
donors or
acceptors, hydrophobic/lipophilic sites, positively ionizable sites, or
negatively
ionizable sites; and
(b) adding geometric constraints to selected mapped features.
2o The fit of the modified test compound may then be evaluated using the same
criteria.
The chemical modification of a group may either enhance or reduce hydrogen
bonding
interaction, charge interaction, hydrophobic interaction, Van Der Waals
interaction or dipole
interaction between the test compound and the keyamino acid residues) of the
binding pocket.
Preferably the group modifications involve the addition, removal, or
replacement of substituents
onto the test compound such that the substituents are positioned to collide or
to bind
preferentially with one or more amino acid residues that correspond to the key
amino acid
residues of the binding pocket.
If a modified test compound model has an improved fit, then it may bind to a
binding
pocket and be considered to be a "ligand". Rational modification of groups may
be made with
3o the aid of libraries of molecular fragments which may be screened for
their: capacity to fit into
CA 02373918 2002-02-28
43
the available space and to interact with the appropriate atoms. Databases of
computer
representations of libraries of chemical groups are available commercially,
for this purpose.
The test compound may also be modified "in situ" (i.e. once docked into the
potential
binding pocket), enabling immediate evaluation of the effect of replacing
selected groups. The
computer representation of the test compound may be modified by deleting a
chemical group or
groups, or by adding a chemical group or groups. After each modification to a
compound, the
atoms of the modified compound and potential binding pocket can be shifted in
conformation
and the distance between the modulator and the binding pocket atoms may be
scored on the basis
of geometric fit and favourable complementary interactions between the
molecules. This
1o technique is described in detail in Molecular Simulations User Manual, 1995
in LUDI.
Examples of ligand building and/or searching computer include programs in the
Molecular Simulations Package (Catalyst), ISISIHOST, ISIS/BASE, and ISIS/DRAW
(Molecular Designs Limited), and UNTTY (Tripos Associates).
The "starting point" for rational ligand design may be a known ligand for a
polo domain.
For example, in order to identify potential modulators of a polo domain or
polo family kinase, in
particular Sak or Plk, a logical approach would be to start with a known
ligand to produce a
molecule which mimics the binding of the ligand. Such a molecule may, for
example; act as a
competitive inhibitor for the true ligand, or may bind so strongly that the
interaction (and
inhibition) is effectively irreversible.
Such a method may comprise the following steps:
(i) generating a computer model of a binding pocket in complex with a ligand;
(ii) replacing one or more groups on the ligand model to produce a modified
ligand;
and
(iii) evaluating the fit of the modified ligand in the binding pocket.
The replacement groups could be selected and replaced using a compound
construction
program which replaces computer representations of chemical groups with groups
from a
computer database, where the representations of the compounds are defined by
structural
coordinates.
In an embodiment, a screening method is provided for identifying a ligand of a
polo
3o domain, in particular a Sak or Plk polo domain, comprising the step of
using the structural
CA 02373918 2002-02-28
coordinates of a substrate or component thereof, defined in relation to its
spatial association with
a binding pocket of the invention, to generate a compound that is capable of
associating with the
binding pocket.
Screening methods of the present invention may be used to identify compounds
or
entities that associate with a molecule that associates with a polo domain, in
particular a Sak or
Plk polo domain.
Test compounds and ligands which are identified using a crystal or model of
the present
invention can be screened in assays such as those well known in the art.
Screening may be for
example in vitro, in cell culture, and/or in vivo: Biological screening assays
preferably centre on
to activity-based response models, binding assays (which measure how well a
compound binds to a
domain), and bacterial, yeast, and animal cell lines (which measure the
biological effect of a
compound in a cell). The assays may be automated for high throughput screening
in which large
numbers of compounds can be tested to identify compounds with the desired
activity. The
biological assay may also be an assay for the binding activity of a compound
that selectively
binds to the binding pocket compared to other proteins.
LIGANDS/COMPOUNDS IDENTIFIED BY SCREENING METHODS
The present invention provides a ligand or compound identified by a screening
method of
2o the present invention. A ligand or compound may have been designed
rationally by using a
model according to the present invention. A ligand or compound identified
using the screening
methods of the invention specifically associate with a target compound, or
part thereof (e.g. a
binding pocket): In the present invention the target compound may be the polo
family kinase
(e.g. Sak or Plk) or part thereof (polo domain), or a molecule that is capable
of associating with
the polo family kinase or polo domain (e.g. substrate).
A ligand or compound identified using a screening method of the invention may
act as a
"modulator", i.e. a compound which affects the activity of a polo family
kinase, in particular
Sak or Plk. A modulator may reduce, enhance or alter the biological function
of a polo family
kinase in particular Sak or Plk. For example a modulator may modulate the
capacity of the
CA 02373918 2002-02-28
enzyme to phosphorylate. An alteration in biological function may be
characterised by a change
in specificity. In order to exert its function, the modulator commonly binds
to a binding pocket.
A "modulator" which is capable of reducing the biological function of the
enzyme may
also be known as an inhibitor. Preferably an inhibitor reduces or blocks the
capacity of the
5 enzyme to phosphorylate. The inhibitor may mimic the binding of a substrate,
for example, it
may be a substrate analogue. A substrate analogue may be designed by
considering the
interactions between the substrate and a polo domain (for example by using
information
derivable from the crystal of the invention) and specifically altering one or
more groups (as
described above)
to The present invention also provides a method for modulating the activity of
a polo family
kinase, in particular Sak or Plk, using a modulator according to the present
invention. It would
be possible to monitor activity following such treatment by a number of
methods known in the
art.
A modulator may be an agonist, partial agonist, partial inverse agonist or
antagonist of a
15 polo family kinase.
As used herein, the term "agonist" means any ligand, which is capable of
binding to a
binding pocket and which is capable of increasing a proportion of the protein
that is in an active
form, resulting in an increased biological response. The term includes partial
agonists and
inverse agonists.
2o As used herein, the term "partial agonist" means an agonist that is unable
to evoke the
maximal response of a biological system, even at a concentration sufficient to
saturate the
specific proteins.
As used herein, the term "partial inverse agonist" is an inverse agonist that
evokes a
submaximal response to a biological- system, even at a concentration
sufficient to saturate the
25 specific proteins. At high concentrations, it will diminish the actions of
a full inverse agonist.
As used herein, the term "antagonist" means any agent that reduces the action
of another
agent, such as an agonist. The antagonist may act at the same site as the
agonist (competitive
antagonism). The antagonistic action may result from a combination of the
substance being
antagonised (chemical antagonism) or the production of an opposite effect
'through a different
3o protein (functional antagonism or physiological antagonism) or as a
consequence of competition
CA 02373918 2002-02-28
46
for the binding site of an intermediate that links enzyme activation to the
effect observed
(indirect antagonism).
As used herein, the term "competitive antagonism" refers to the competition
between an
agonist and an antagonist for a protein that occurs when the binding of
agonist and antagonist
becomes mutually exclusive. This may be because the agonist and antagonist
compete for the
same binding sites or combine with adjacent but overlapping sites. A third
possibility is that
different sites are involved but that they influence the protein
macromolecules in such a way that
agonist and antagonist molecules cannot be bound at the same time. If the
agonist and antagonist
form only short lived combinations with the protein so that equilibrium
between agonist,
antagonist and protein is reached during the presence of the agonist, the
antagonism will be
surmountable over a wide range of concentrations. In contrast, some
antagonists, when in close
enough proximity to their binding site, may form a stable covalent bond with
it and the
antagonism becomes insurmountable when no spare proteins remain.
As mentioned above, an identified ligand or compound may act as a ligand model
(for
example, a template) for the development of other compounds. A modulator may
be a mimetic
of a ligand.
Like the test compound (see above) a modulator may be one or a variety of
different sorts
of molecule.(See examples herein.) A modulator may be an endogenous
physiological
compound, or it may be a natural or synthetic compound. The modulators of the
present
invention may be natural or synthetic. The term "modulator" also refers to a
chemically
modified ligand or compound.
The technique suitable for preparing a modulator will depend on its chemical
nature. For
example, peptides can be synthesized by solid phase techniques (Roberge JY et
al (1995)
Science 269: 202-204) and automated synthesis may be achieved, for example,
using the ABI 43
1 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions
provided by the
manufacturer. Once cleaved from the resin; the peptide may be purified by
preparative high
performance liquid'chromatography (e.g., Creighton (1983) Proteins Structures
and Molecular
Principles, WH Freeman and Co, New York NY). The composition of the synthetic
peptides
may be confirmed by amino acid analysis or sequencing (e.g., the Edman
degradation procedure;
Creighton, supra).
CA 02373918 2002-02-28
47
Organic compounds may be prepared by organic synthetic methods described in
references such as March, 1994, Advanced Organic Chemistry: Reactions,
Mechanisms, and
Structure, New York, McGraw Hill:
The invention also relates to classes of modulators of polo family kinases
based on the
structure and shape of a substrate or component thereof, defined in relation
to the substrate's
spatial association with a crystal structure of the invention or part thereof.
The invention contemplates all optical isomers and racemic forms of the
modulators of
the invention.
io COMPOSITIONS
The present invention also provides the use of a modulator according to the
invention, in
the manufacture of a medicament to reat and/or prevent a disease in a
mammalian patient.
There is also provided a pharmaceutical composition comprising such a
modulator and a method
of treating and/or preventing a disease comprising the step of administering
such a modulator or
composition to a mammalian patient
The pharmaceutical compositions may be for human or animal usage in human and
veterinary medicine and will typically comprise a pharmaceutically acceptable
carrier; diluent,
excipient, adjuvant or combination thereof.
Acceptable earners or diluents for therapeutic use are well known in the
pharmaceutical
art, and are described, for example, in Remington's Pharmaceutical Sciences,
Mack Publishing
Co. (A. R. Gennaro edit. 1985). The choice of pharmaceutical carrier,
excipient or diluent can
be selected with regard to the intended route of administration and standard
pharmaceutical
practice. The pharmaceutical compositions may also comprise suitable
binder(s), lubricant(s),
suspending agent(s), coating agent(s), solubilising agent(s).
Preservatives, stabilizers, dyes and even flavouring agents may be provided in
the
pharmaceutical composition. Examples of preservatives include sodium benzoate,
sorbic acid
and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be
also used.
The routes for administration (delivery) include, but are not limited to, one
or more of:
oral (e.g. as a tablet, capsule, or as an ingestable solution), topical,
mucosal (e.g. as a nasal spray
CA 02373918 2002-02-28
48
or aerosol for inhalation), nasal, parenteral (e.g. by an injectable form),
gastrointestinal,
intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine,
intraocular, intradermal,
intracranial, intratracheal, intravaginal, intracerebroventricular,
intracerebral, subcutaneous,
ophthalmic (including intravitreal or intracameral); transdermal, rectal,
buccal, vaginal, epidural,
sublingual.
Where the pharmaceutical composition is to be delivered mucosally through the
gastrointestinal mucosa; it should be able to remain stable during transit
though the
gastrointestinal tract; for example, it should be resistant to proteolytic
degradation, stable at acid
pH and resistant to the detergent effects of bile.
l0 Where appropriate, the pharmaceutical compositions can be administered by
inhalation,
in the form of a suppository or pessary, topically in the form of a lotion,
gel; hydrogel, solution,
cream, ointment or dusting powder, by use of a skin patch, orally in the form
of tablets
containing excipients such as starch or lactose or chalk, or in capsules or
ovules either alone or in
admixture with excipients, or in the form of elixirs, solutions or suspensions
containing
flavouring or colouring agents, or they can be injected parenterally, for
example, intravenously,
intramuscularly or subcutaneously. For parenteral administration, the
compositions may be best
used in the form of a sterile aqueous solution which may contain other
substances, for example
enough salts or monosaccharides to make the solution isotonic with blood. The
aqueous solutions
should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
The preparation of
suitable parenteral formulations under sterile conditions is readily
accomplished by standard
pharmaceutical techniques well-known to those skilled in the art.
If the agent of the present invention is administered parenterally, then
examples of such
administration include one or more of: intravenously, intra-arterially,
intraperitoneally,
intrathecally, intraventricularly, intraurethrally, intrasternally,
intracranially, intramuscularly or
subcutaneously administering the agent; and/or by using infusion techniques.
For buccal or sublingual administration the compositions may be administered
in the
form of tablets or lozenges which can be formulated in a conventional manner.
The tablets may contain excipients such as microcrystalline cellulose,
lactose, sodium
citrate, calcium carbonate, dibasic calcium phosphate and glycine,
disintegrants such as starch
(preferably corn, potato or tapioca starch), sodium starch glycollate,
croscarmellose sodium and
CA 02373918 2002-02-28
49
certain complex silicates, and granulation binders such as
polyvinylpyrrolidone,
hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose,
gelatin and
acacia. Additionally, lubricating agents such as magnesium stearate, stearic
acid, glyceryl
behenate and talc may be included.
Solid compositions of a similar type may also be employed as fillers in
gelatin capsules.
Preferred excipients in this regard include'lactose, starch; cellulose, milk
sugar or high molecular
weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent
may be
combined with various sweetening or flavouring agents, colouring matter or
dyes, with
emulsifying and/or suspending agents and with diluents such as water, ethanol,
propylene glycol
to and glycerin, and combinations thereof.
As indicated, the therapeutic agent (e.g. modulator) of the present invention
can be
administered intranasally or by inhalation and is conveniently delivered in
the form of a dry
powder inhaler or an aerosol spray presentation from a pressurised container,
pump, spray or
nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as
1,1,1,2-
tetrafluoroethane (HFA 134A~) or 1,1,1,2,3,3,3-heptafluoropropane (HFA
227EA~), carbon
dioxide or other suitable gas. In the case of a pressurised aerosol, the
dosage unit may be
determined by providing a valve to deliver a metered amount. The pressurised
container, pump,
spray or nebuliser may contain a solution or suspension of the active
compound, e.g. using a
mixture of ethanol and the propellant as the solvent; which may additionally
contain a lubricant,
e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from
gelatin) for use in an
inhaler or insufflator may be formulated to contain a powder mix of the went
and a suitable
powder base such as lactose or starch.
Therapeutic administaration of polypeptide modulators may also be accomplished
using
gene therapy. A nucleic acid including a promoter operatively linked to a
heterologous
polypeptide may be used to produce high-level expression of the polypeptide in
cells transfected
with the nucleic acid: DNA or isolated nucleic acids may be introduced into
cells of a subject by
conventional nucleic acid delivery systems. Suitable delivery systems include
liposomes, naked
DNA, and receptor-mediated delivery systems, and viral vectors such as
retroviruses, herpes
viruses, and adenoviruses.
CA 02373918 2002-02-28
The invention further provides a method of treating a mammal, the method
comprising
administering to a mammal a modulator or pharmaceutical composition of the
present invention.
Typically, a physician will determine the actual dosage which will be most
suitable for an
individual subject and it will vary with the age, weight and response of the
particular patient and
5 severity of the condition. The dosages below are exemplary of the average
case. There can, of
course, be individual instances where higher or lower dosage ranges are
merited.
The specific dose level and frequency of dosage for any particular patient may
be varied
and will depend upon a variety of factors including the activity of the
specific compound
employed, the metabolic stability and length of action of that compound, the
age, body weight,
10 general health, sex,, diet, mode and time of administration, rate of
excretion, drug combination,
the severity of the particular condition; and the individual undergoing
therapy. By way of
example; the pharmaceutical composition of the present invention may be
administered in
accordance with a regimen of 1 to 10 times per day, such as once or twice per
day.
For oral and parenteral administration to human patients, the daily dosage
level of the
15 agent may be in single or divided doses.
APPLICATIONS
The modulators and compositions of the invention may be useful in treating,
inhibiting,
or preventing diseases modulated by polo family kinases. They may be used to
treat, inhibit, or
20 prevent proliferative diseases. The modulators may be used to stimulate or
inhibit cell
proliferation.
Accordingly, modulators of the invention may be useful in the prevention and
treatment
of conditions including but not limited to lymphoproliferative conditions,
malignant and pre-
malignant conditions, arthritis, inflammation, and autoimrnune disorders.
Malignant and pre-
25 malignant conditions may include solid tumors, B cell lymphomas, chronic
lymphocytie
leukemia, chronic myelogenous leukemia, prostate hypertrophy, HirSchsprung
disease,
glioblastoma, breast and ovarian cancer, adenocarcinoma of the salivary gland,
premyelocytic
leukemia, prostate cancer, multiple endocrine neoplasia type IIA and IIB,
xnedullary thyroid
carcinoma; papillary carcinoma, papillary renal carcinoma, hepatocellular
carcinoma,
30 gastrointestinal stromal tumors, sporadic mastocytosis, acute myeloid
leukemia, large cell
CA 02373918 2002-02-28
51
lymphoma or Alk lymphoma; chronic myeloid leukemia, hematological /solid
tumors, papillary
thyroid carcinoma, stem cell leukemiallymphoma syndrome, acure myelogenous
lleukemia,
osteosareoma, multiple myeloma, preneoplastic liver foci, and resistance to
chemotherapy.
Diseases associated with increased cell survival, or the inhibition of
apoptosis, include cancers
(e.g. follicular lymphomas, carcinomas with p53 mutations, hormone-dependent
tumors such as
breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer);
autoimmune disorders
(such as lupus erythematosus and immune-related glomerulonephritis rheumatoid
arthritis) and
viral infections (such as herpes viruses, pox viruses, and adenoviruses);
inflammation, graft vs.
host disease, acute graft rejection and chronic graft rejection:
to Modulators that stimulate cell proliferation may be useful in the treatment
of conditions
involving damaged cells including conditions in which degeneration of tissue
occurs such as
arthropathy, bone resorption, inflammatory disease, degenerative disorders of
the central nervous
system, and for promoting wound healing.
The invention will now be illustrated by the following non-limiting examples:
is EXAMPLES:
Example 1
The following methods were used in the investigation described in the example:
Protein expression, mutagenesis and purification: The C-terminus of Sak-a
encompassing the
polo domain (residues 839 to 925) was expressed in E. coli as a GST fusion
protein using the
20 pGEX-2T vector (Pharmacia). The Quickchange kit (Stratagene) was used to
generate site
directed va1874met mutants for heavy atom phasing. Protein was purified by
affinity
chromatography using glutathione Sepharose beads (Pharmacia). Bound protein
was eluted by
cleavage with thrombin. Eluate was applied to HiQ ion-exchange column under
low salt
conditions. The flow through containing protein was concentrated to
approximately 1mM and
25 was applied to a Superdex 75 gel filtration column (Pharmacia) for final
purification and
characterization.
Crystallization and data collection: Hanging drops containing lpl of 50 mg/ml
native or
mutant protein in 20mM Hepes pH 8.0, SmM DTT, were mixed with equal volumes of
reservoir
buffer containing 100mM Tris pH 7.0, 32.5% (v/v) Jeffamine M-600 (Hampton);
and 200mM
3o MgCI. Hexagonal like crystals of approximate dimensions 0.10 x 0.10 x 0.03
mm were obtained
CA 02373918 2002-02-28
52
overnight. The crystals contain two molecules of the Sak polo domain per
asymmetric unit, and
belong to the space group P3212;, (a = b _ 51.782 A, c _ 146.941 A). The
solution dimer
corresponds to the NCS related dimer of asymmetric unit.
MAD diffraction data was collected on frozen crystals at Structural Biology
Center 19
BM and BIOCARS 14 BMC at the Advanced Photon Source in Argonne National
Laboratory.
Data processing and reduction was carried out using HKL 2000. Heavy atom sites
were
identified using CNS and protein phases were calculated using SHARP.
Model building and Refinement: Model building was performed using O (Jones,
1991). A
starting model comprising approximately 65% of the total structure was refined
using CNS. Bulk
to solvent correction was applied during refinement and simulated annealing
protocols were
employed. The remaining structure was built into 2Fo F~ electron density maps
generated with
CNS. The final refinement statistics are shown in Table 1. The first 7 and
last 6 residues of the
polo domain fragment are disordered (residues 839 to 845 and residues 920 to
925) and have not
been modeled. No amino acid residues occupy disallowed regions of the
Ramachandran plot and
94% occupy the most favored regions.
Sak protein localization: To generate pEGFP-Sak, a murine Sak cDNA construct,
pCD-HA-
Sak was digested with EcoRI and ApaI and the cDNA fragment ligated into pEGFP-
C1
(Clontech) resulting in a construct with an N-terminal in frame fusion of GFP
onto HA-Sak.
Similarly, the polo domain of Sak, amino acids 826-925, was placed in frame
with GFP by
2o excising a BamHI and EcoRI from Sak; and ligating into the BgIII and EcoRI
sites of pEGFP-C1
to produce pEGFP-pb. An ApaI site was introduced into the Sak cDNA sequence at
nt 2670 by
PCR based site directed mutagenesis (Stratagene), the polo domain was removed
by digestion
with ApaI and re-ligated of the plasmid to generate pEGFP-SakOpb. To generate
a tetracycline
inducible Sak expression construct, pEGFP-Sak was linearized with ApaI, end-
filled with
Klenow and digested with Eco47III: The resultant GFP-Sak fragment was ligated
into an end-
filled BamHI site of pSTAR.
NIH 3T3 murine fibroblast cells were maintained in DMEM containing 10% FCS.
For
transient gene expression, cells at 20-30% confluence on glass cover slips
were transfected with
1 pg of plasmid DNA, pEGFP- Sak, pEGFP-SakOpb; pEGFP-pb or pEGFP-C1 with
EffecteneT""
(Qiagen). Cell lines expressing GFP-Sak and GFP were generated by stably
transfecting NIH-
CA 02373918 2002-02-28
53
3T3 cells with the tetracycline inducible construct pSTAR-GFP-Sak and pSTAR-
GFP, selecting
cells in 6418, followed by induction of transgene expression with 2 ~.g/mL
doxycycline for 6
hours. GFP-expressing cells were sorted by FAGS and cloned by limiting
dilution.
Stable and transiently transfected NIFi-3T3 cells on glass cover-slips were
released from
36 hr of serum starvation by addition of fresh media containing 10% FBS,
either with or without
doxycycline at 2 ~.g/mL, and fixed at intervals as they proceed through the
cell cycle. The
coverslips were processed by rinsing twice briefly in PBS, permeabilizing for
5 min in PBS 0.5%
Triton X-100, then fixed in 3.7% para-formaldehyde/PBS for 12 minutes. Actin
nucrofilaments
were stained with a 1:100 dilution of TRITC-phalloidin (Sigma) in PBS. Anti-y
tubulin antibody
(Sigma) was used at 1:200 dilution in Tris/Saline 0:1% Tween20 at 20°C
for 30 min. Cells were
washed three times in Tris/Saline 0.1% Tween20 and incubated in 1:500 dilution
of rhodamine-
conjugated goat anti-mouse secondary antibody for 20 minutes. Nuclei were
stained with
Hoechst 33258 in PBS for 20 secopds. Images were obtained using an Olyrr~pus
IX-70 inverted
microsope equipped with a Princeton CCD camera and Deltavision Deconvolution
microscopy
software (Applied Precision). Transiently transfected and stable cell lines
gave identical patterns
of localization. Images of GFP-Sak expression are taken from cell lines
containing the pSTAR-
GFP-Sak vector, and all other images are from transiently transfected cells.
Immmunoprecipitation:
NIH 3T3 murine fibroblast cells were maintained in DMEM containing 10% FCS.
For
2o transient gene expression, cells at 30 to 40% confluence were transfected
using EffecteneTM
(Qiagen): Cells were lysed in 50mM Tris pH 7.5, 150mM NaCI, 1mM EDTA, 0.5%
Triton-X
100. Immunoprecipitations were performed using anti-flag antibody (Sigma) and
Protein G
Sepharose (Pharmacia) according to product specifications. Beads were washed
three times with
50mM Tris pH 7.5, 150mM NaCI, Q.1 % Triton-X 100. Immunoprecipitations were
blotted with
1:200 dilution of anti-myc antibody (Santa-Cruz Biotech) or 1:4000 dilution of
anti-flag antibody
(Sigma).
Results and Discussison
A protein fragment encompassing the polo box motif of Sak (residues 839 to
925) was
expressed and characterized. Using limited proteolysis and mass spectrometry,
a protease
resistant domain was identified that behaves as a dimer in solution as
indicated by static light
CA 02373918 2002-02-28
54
scattering analysis and size exclusion chromatography (Figure 2). This domain
was crystallized
(Figure 3) and its structure was determined using the selenomethione-multiple
anomalous
dispersion (SeMet MAD) technique (see methods). Structure determination and
crystallographic
refinement statistics are provided in Table 1.
The crystal structure of the polo domain of Sak is dimeric in nature,
consisting of a two-
sheet, strand-exchange (3-fold (Figure 4ab). Analysis by VAST and DALI
identifies this
structure as a novel protein fold. The topology of the monomer consists from
its amino terminus
of five (3-strands ((3i-(35), one oc-helix (ocA),.and a C-terminal (3-strand
((36). (3-strands 6; l, 2, and
3 from one monomer form a contiguous anti parallel sheet with (3-strands 4 and
5 from the
1o second monomer: The two (3-sheets pack with a crossing angle of
110°, orienting hydrophobic
surfaces inwards and hydrophilic surfaces outwards. Helix ocA, which is
collinear with (3-strand
6 of the same monomer, buries a large portion of the non-overlapping
hydrophobic (3-sheet
surfaces. Interactions involving helices aA compose a majority of the
hydrophobic core structure
and also the dimer interface. The total surface area buried by dimer formation
is 2447.78 A2.
Overall, the dimeric structure is clam like (60A x 44A x 20A), hinged at one
end through the
seamless association of ~3-strands 3 from each monomer (Figure 4b). Extending
inwards from
the mouth of the structure is a deep cavity of approximate dimensions 17A x
8.5~ x 11.3A
(Figure 5a-c). The entranceway to this cavity is relatively small (17A x 7.5A)
and is partitioned
in two by the contact of the Trp 853 side chains from each polypeptide of the
dimer.
The hydrophobic nature of the dimer interface is highly conserved across the
Sak
orthologues and also the more distantly related polo family members (see
alignment Figure 1).
Of 21 conserved hydrophobic positions, 13 participate in dimes formation. The
mutation of one
dimes interface position in Plk, analogous to Leu 857 to Ala in Sak, has been
shown to abolish
Plk's ability to complement the cdc5 temperature ensitive phenotype in yeast.
Interestingly,
only two charged residues are conserved amongst the different polo domain
sequences, namely
Asp 868 and Lys 906 in Sak, and these residues form a salt interaction across
the dimes interface.
Together these observations suggest that the dimes configuration revealed by
the crystal structure
may be biologically relevant and that the polo domain of other family members
may form similar
dimeric (3-sheet structures.
CA 02373918 2002-02-28
The presence of two polo box motifs in most polo family kinases raises the
possibility for
both intramolecular and intermolecular modes of molecular dimerization. A
striking covariance
across the paired polo box motifs suggests that polo box one and two may
associate
preferentially (either in an intramolecular or intermolecular manner). For
example, Val 846 in
5 Sak, which locates to the dimer interface, is substituted with aspartic acid
in the first but not the
second polo box motif of all tandemly repeated family members. The
introduction of a charged
side chain into the hydrophobic dimer interface region appears to be
accommodated by the
substitution of Lys 906 with Arg in the second polo box motif at the
presumptive hetero-dimer
interface: The two positions while distant in primary structure locate to
within 5 angstroms in
10 the crystal structure. Substituting Lys 906 with arginine and Val 846 with
aspartic acid would
bring these residues to within 3 angstroms in the crystal structure. As
position 906 is already
committed to a salt interaction with Asp 868, the increased interaction
potential of the
guanidinium group of Arginine may allow for the formation of a dual salt
interaction.
Suggesting that an intramolecular mode of dimerization is feasible, the linker
region between
15 tandem polo box motifs of all family members is sufficiently long (19 to 34
amino acids) to
bridge the 36A distance separating the amino and carboxy termini of opposing
dimer chains in
the crystal structure.
Next to the dimer interface region, the interfacial cleft region is the most
conserved part
of the polo domain structure. When the polo domain sequences of Fnk/Prk, Snk,
and Plk are
2o modeled onto the Sak crystal structure, the approximate dimensions and
characteristics of the
pocket and cleft region are also well preserved. Additional polo domain
mutants in plk and cdc5
have been characterized for localization activity and complementation of the
cdc5-1 temperature
sensitive mutation (Lee et al, 1998)(Song et al., 2000). Substitutions of Trp
414 with Phe or Val
415 with Ala in Plk, analogous to Lys 844 and Ser 8:45 in the Sak polo domain
primary structure,
25 are located immediately adjacent to the interfacial cleft region. In
addition; a substitution of the
conserved residue Asn 437 to Asp in Plk corresponds to the highly conserved
interfacial cleft
residue Asn 867 in the crystal structure. Based on these observations, the
interfacial cleft and
pocket region may function as a ligand-binding site that localizes the protein
to mitotic
structures.
CA 02373918 2002-02-28
56
To investigate the role of the polo domain in the subcellular localization of
Sak, GFP
fusion constructs of Sak, SakOpb and the polo domain were transiently
transfected into NIH 3T3
cells and examined using immunofluorescence. GFP-Sak colocalizes with y-
tubulin and actin,
indicating the position of the centrosomes and cleavage furrow respectively.
This localization
pattern has also been observed upon overexpression of Plk (Golsteyn et al,
1995). The polo
domain itself is sufficient for localization to the centrosomes and cleavage
furrow. Concurrent
with observations by Jang et al. when investigating the subcellular
localization of the C-terminus
of Plk (Jang et al., 2002), localization of the polo domain is observed with
low expression levels,
while cells with increased expression exhibit strong diffuse cytoplasmic
staining. Deletion of the
to polo domain does not abolish subcellular localization, however may arise
from kinase domain
dependent interaction with endogenous Sak.
The polo domain is a dimeric structure composed of two polo box motifs, and a
putative
interaction surface was identified that mediates the domains localization
function. Recent
reports using two hybrid and imrnunoprecipitation experiments suggest that the
polo domain of
CdcS binds directly with septins which themselves localize to the cleavage
furrow at cytokinesis.
The binding surface of Sak looks very different from other peptide binding
surfaces
characterized to date. Rather than a large flat face like SH2, SH3 and WW
domains, it appears
more similar to the dimeric 14-3-3 domains, which have semi shielded binding
pockets.
Difference in localization of Sak and other polo family kinases also suggest
multiple binding
partners for the pb motifs. The polo domain of Sak but not Plk localizes to
the nucleolus (Hudson
2001) while Plk but not Sak localizes to centromeres and the midbody at
metaphase [{Arnaud,
Pines, et al. 1998 } {Wianny, Tavares, et al. 1998) {Logarinho & Sunkel
1998}]: In addition,
over expressed Snk also localizes to non mitotic membrane structures the
significance of which
is not understood (Holtrich 2000, Kauseimann, 1999). While the polo domains
are predicted to
adopt very similar dimeric structures; differences in amino acid sequences are
considerable even
in the more conserved dimes interface. If polo domains do have different
binding partners and
localization specificity, this may reflect a specialization and divergence of
polo family functions
in higher eukaryotes.
Mutation of Sak in ES cells by gene targeting has demonstrated the requirement
for the
enzyme, as Sak null embryos arrest development at E7.5 with cells delayed in
late mitosis and
CA 02373918 2002-02-28
57
increased apoptotic death. Either overexpression or loss of Sak causes lass of
cell viability and
toxicity of the former is dependent on the presence of the polo domain. Plk
has been shown to
be a potential therapeutic target as over expression of a kinase-deficient
form of Plk results in
cell death, an apparent dominant-negative. effect which is more pronounced' in
tumor cells than
non-transformed cells. These observations suggest the pola like kinases may be
potential targets
for intervention to treat cell proliferative diseases such as cancer where
check point controls are
generally deficient. Herein, is described a dimeric structure of the polo
domain, and an
interfacial cleft region that is required to localize enzymes to mitotic
structures. Unlike the S/T-
kinase domain, the polo domain is unique to this small family of enzymes that
regulate mitotic
1o progression. Other attractive features for intervention include a large
semi-enclosed pocket for
engineering binding and selectivity properties, a solvent accessible site for
the engineering of
pharmaco-kinetic properties. Because there are significant differences between
polo domains of
different members there is also the potential to engineer specificity into the
drugs:
The present invention is not to be limited in scope by the specific
embodiments described
herein, since such embodiments are intended as but single illustrations of one
aspect of the
invention and any functionally equivalent embodiments are within the scope of
this invention.
Indeed, various modifications of the invention in addition to those shown and
described-herein
will become apparent to those skilled in the art from the foregoing
description and accompanying
drawings. Such modifications are intended to fall within the scope of the
appended claims.
A11 publications, patents and patent applications referred to herein are
incorporated by
reference in their entirety to the same extent as if each individual
publication, patent or patent
application was specifically and individually indicated to be incorporated by
reference in its
entirety. All publications, patents and patent applications mentioned herein
are incorporated
herein by reference for the purpose of describing and disclosing the cell
lines, vectors,
methodologies etc. which are reported therein which might be used in
connection with the
invention. Nothing herein is to be construed as an admission that the
invention is not entitled to
antedate such disclosure by virtue of prior invention:
CA 02373918 2002-02-28
58
It must be noted that as used herein and in the appended claims, the singular
forms "a",
"an", and "the" include plural reference unless the comext clearly dictates
otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such host cells,
reference to the
"antibody" is a reference to one or more antibodies and equivalents thereof
known to those
skilled in the art; and so forth.
CA 02373918 2002-02-28
59
.
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CA 02373918 2002-02-28
Table 2
REMARKcoordinates from
minimization
and B-factor
refinement
REMARKrefinement resolution:
500.0 - 2.0
A
REMARKstarting r= 0.2341
freer= 0.2471
REMARKfinal r= 0.2312
freer= 0.2489
10 REMARKrmsd bonds= 0.011680
rmsd angles=
1.53873
REMARKB rmsd for bonded
mainchain atoms=
1.546 target=
1.5
REMARKB rmsd for bonded
sidechain atoms=
2.297 target=
2.0
REMARKB rmsd for angle
mainchain atoms=
2.242 target=
2.0
REMARKB rmsd for angle
sidechain atoms=
3.450 target=
2.5
15 REMARKtarget= mlf final
wa= 2.67
REMARKfinal rweight=
0.1829 (with
wa= 2.67)
REMARKmd-method= torsion
annealing schedule=
slowcool
REMARKstarting temperature=
600 total and
steps= 6 * 6
REMARKcycles= 2 coordinate
steps= 20' B-factor
steps= 10
20 REMARKsg= P3(2)12 a= eta= 90 gamma=
51:782 b= 51.782
c = 146.941
alpha= 90 b
120
REMARKtopology file
1 . CNS TOPPAR:protein.top
REMARKtopology file
2 CNS TOPPAR:dna-rna.top
REMARKtopology file
3 . CNS TOPPAR:wa~er.top
25 REMARKtopology file
4 . CNS TOPPAR:ion.top
REMARKparameter file
1 . CNS TOPPAR:protein
rep.param
REMARKparameter file
2 . CNS_TOPPAR:dna-rna
rep.param
REMARKparameter file
3 GNS_TOPPAR:water_rep.param
REMARKparameter file
4 CNS_TOPPAR:ion.param
30 REMARKmolecular structure
file: automatic
REMARKinput coordinates.
refine28.pdb
REMARKreflection file=
peakl:cv
REMARKncs= none
REMARKB-correction
resolution:
6.0 - 2.0
35 REMARKinitial B-factor
correction applied
to fobs
REMARKB11= -1.018 B22=
-1.018 B33=
2.036
REMARKB12= -3.420 B13=
O.OOO B23= 0.000
REMARKB-factor correction 1'.378
applied to coordinate
array B: -
REMARKbulk solvent: 59.3227 A~2
density level=
0.389731 e/A~3,
B-factor=
40 REMARKreflections withFobs/sigma_F < 0.0 rejected
REMARKreflections withFobs> 10000 * rms(Fobs) rejected
REMARKanomalous diffraction ata was input
d
REMARKtheoretical total 29811 ( 100.0
) number of refl: ~
in resol. range:
45 REMARKnumber of unobserved 537 ( 1.8 ~
reflections
(no entry or
~F~=0):
REMARKnumber of reflections 0 ( 0.0 $
rejected:
REMARKtotal number 29274 ( 98.2
of reflections ~
used:
50 )
REMARKnumber of reflections 26435 ( 88.7
in working set: ~
REMARKnumber of reflections 2839 ( 9.5
in test set: ~
55 CRYST151.782 51.782 2
14.6.941 90.00
90.00 120.00
P 32 1
REMARKFILENAME="refine29.pdb"
REMARKDATE:11-Jan-01
15:10:17 created
by user: leung
REMARKVERSION:1.0
ATOM 1 CB SER A 8 48.49 A
18.661 18.360
26.264 1.00
60 ATOM 2 OG SER A 8 50.47 A
19.163 19.370
27.127 1.00
ATOM 3 C SER A 8 16.981 45.01 A
16.981 27.538
1.00
CA 02373918 2002-02-28
61
ATOM 4 0 SERA 8 16.148 16.296 26.9401.00 45.75 A
ATOM 5 N SERA 8 18.698 15.879 26.1531.00 47.55 A
ATOM 6 CA SERA 8 T8.430 17.054 27.0401.00 47.07 A
ATOM 7 N VALA 9 16.678 17.677 28.6291.00 41.83 A
ATOM 8 CA VALA 9 15.323 17.661 29.1721.00 38.86 A
ATOM 9 CB VALA 9 15.355 17.713 30.7201.00 39.15 A
ATOM 10 CG1 VALA 9 16.094 18.970 31.1811.00 40.82 A
ATOM 11 CG2 VALA 9 13.937 17.705 31.2801.00 39.80 A
ATOM 12 C VALA 9 14.511 18.853 28.6411.00 36.67 A
ATOM 13 0 VALA 9 15.001 19.985 28.5911.00 35.40 A
ATOM 14 N PHEA 10 13.280 18.603 28.2161.00 33.75 A
ATOM 15 CA PHEA 10 12.457 19.698 27.7401.00 33.36 A
ATOM 16 CB PHEA 10 12.733 19.956 26.2421.00 36.21 A
ATOM 17 CG PHEA 10 12.536 18.753 25.3661.00 38.49 A
ATOM 18 CD1 PHEA 10 11.284 18.460 24.8341.00 39.36 A
ATOM 19 CD2 PHEA 10 13.590 17.888 25.1051.00 40.33 A
ATOM 20 CE1 PHEA 10 11.083 17.326 24.0591.00 38.97 A
ATOM 21 CE2 PHEA 10 13.393 16.740 24.3231.00 40.13 A
ATOM 22 CZ PHEA 10 12.137 16.461 23.8021.00 39.15 A
ATOM 23 C PHEA 10 10.992 19.393 28.0021.00 31.66 A
ATOM 24 0 PHEA 10 10.615 18.234 28.2241.00 29.02 A
ATOM 25 N VALA 11 10.170 20.442 28.0161.00 28.88 A
ATOM 26 CA VALA 11 8.731 20.291 28.2391.00 28.95 A
ATOM 27 CB VALA 11 8.050 21.686 28.4171.00 29.73 A
ATOM 28 CG1 VALA 11 6.538 21.516 28.6431.00 29.60 A
ATOM 29 CG2 VALA 11 8.678 22.417 29.6011.00 27.93 A
ATOM 30 C VALA 11 8.109 19.572 27.0411.00 29.58 A
ATOM 31 0 VALA 11 8.447 19.861 25.8961.00 31.80 A
ATOM 32 N LYSA 12 7.215 18.633 27.2951.00 28.97 A
ATOM 33 CA LYSA 12 6.568 17.898 26.2231.00 29.28 A
ATOM 34 CB LYSA 12 6.699 16.405 26.4851.00 31.10 A
ATOM 35 CG LYSA 12 5.974 15.520 25.5171.00 34.59 A
ATOM 36 CD LYSA 12 6.087 14.074 25.9811.00 38:87 A
ATOM 37 CE LYSA 12 5.482 13.130 24.9511.00 42.52 A
ATOM 38 NZ LYSA 12 5.795 11.708 25.2741.00 45.41 A
ATOM 39 C LYSA 12 5.076 18.286 26.1531.00 28.89 A
ATOM 40 0 LYSA 12 4.551 18.561 25.0621.00 28.53 A
ATOM 41 N ASNA 13 4.406 18.293 27.3101.00 26.75 A
ATOM 42 CA ASNA 13 2.983 18.663 27.3981.00 25.69 A
ATOM 43 CB ASNA 13 2.066 17.447 27.5941.00 25.72 A
ATOM 44 CG ASNA 13 2.329 16.336 26.5951.00 28.05 A
ATOM 45 OD1 ASNA 13 2.591 16.596 25.4161.00 28.45 A
ATOM 46 ND2 ASNA 13 2.234 15.088 27.0531.00 26.70 A
ATOM 47 C ASNA 13 2.798 19.568 28.6031.00 26.66 A
ATOM 48 0 ASNA 13 3.458 19.381 29.6401.00 24.38 A
ATOM 49 N VALA 14 1.891 20.544 28.4711.00 27.40 A
ATOM 50 CA VALA 14 1.570 21.488 29.5471.00 27.04 A
ATOM 51 CB VALA 14 2.240 22.862 29.3551.00 29.56 A
ATOM 52 CG1 VALA 14 1.802 23.808 30.4631.00 30.90 A
ATOM 53 CG2 VALA 14 3.722 22.735 29.4121.00 29.82 A
ATOM 54 C VALA 14 0.064 21.728 29.5561.00 27.95 A
ATOM 55 0 VALA 14 -0.604 21.699 28.5051.00 25.07 A
ATOM 56 N GLYA 15 -0.476 21.964 30.7431.00 27.09 A
ATOM 57 CA GLYA 15 -1.895 22.227 30.8511.00 25.98 A
ATOM 58 C GLYA 15 -2.156 23.066 32.0831.00 25.20 A
ATOM 59 0 GLYA 15 -1.290 23.206 32.9571.00 23.65 A
ATOM 60 N TRPA 16 -3.333 23.666 32.1501.00 23.21 A
ATOM 61 CA TRPA 16 -3.667 24.451 33.3191.00 22.53 A
ATOM 62 CB TRPA 16 -3.016 25.848 33.2841.00 22.44 A
ATOM 63 CG TRPA 16 -3.597 26.857 32.3021.00 26.17 A
ATOM 64 CD2 TRPA 16 -2.857 27.662 31.3731.00 27.23 A
ATOM 65 CE2 TRPA 16 -3.782 28.549 30.7531.00 28.23 A
CA 02373918 2002-02-28
62
ATOM 66 CE3 TRPA 16 -1.507 27.720 31.0001.00 29.82 A
ATOM 67 CD1 TRPA 16 -4.908 27.275 32.2061.00 25.56 A
ATOM 68 NE1 TRPA 16 -5.020 28.298 31.2781.00 26.09 A
ATOM 69 CZ2 TRPA 16 -3.380 29.490 29.7881.00 30.09 A
ATOM 70 CZ3 TRPA 16 -1.112 28.666 30.0331.00 30.90 A
ATOM 71 CH2 TRPA 16 -2.047 29.531 29.4421.00 29.35 A
ATOM 72 C TRPA 16 -5.153 24.578 33.4181.00 21.37 A
ATOM 73 0 TRPA 16 -5.898 24.354 32.4371.00 19.90 A
ATOM 74 N ALAA 17 -5.607 24.930 34.6141.00 21.50 A
ATOM 75 CA ALAA 17 -7.029 25.121 34.8101.00 21.42 A
ATOM 76 CB ALAA 17 -7:662 23.858 35.3401.00 19.91 A
ATOM 77 C ALAA 17 -7.040 26.193 35.8501.00 22.81 A
ATOM 78 0 ALAA 17 -6.495 25.978 36.9361.00 2'1.78 A
ATOM 79 N THRA 18 -7.623 27.349 35.5191.00 21.72 A
ATOM 80 CA THRA 18 -7.675 28.458 36.4621.00 2'4.80 A
ATOM 81 CB THRA 18 -6.944 29.715 35:9171.00 26.48 A
ATOM 82 OG1 THRA 18 -7.603 30.182 34.7311.00 25.58 A
ATOM 83 CG2 THRA 18 -5.474 29.377 35.5631.00 27.63 A
ATOM 84 C THRA 18 -9.110 28.850 36.8131.00 26.36 A
ATOM 85 0 THRA 18 -10.050 28.600 36.0541.00 25.24 A
ATOM 86 N GLNA 19 -9.265 29.438 37.9901.00 28.51 A
ATOM 87 CA GLNA 19 -10.561 29.886 38.4731.00 31.86 A
ATOM 88 CB GLNA 19 -10.869 29.229 39.8041.00 33.51 A
ATOM 89 GG GLNA 19 -10.742 27.730 39.7621.00 37.86 A
ATOM 90 CD GLNA 19 -11.609 27.085 40.8171.00 43.09 A
ATOM 91 OE1 GLNA 19 -12.846 27.247 40.8021.00 44.57 A
ATOM 92 NE2 GLNA 19 -10.978 26.359 41.7571.00 43.84 A
ATOM 93 C GLNA 19 -10.471 31.399 38.6341.00 33.17 A
ATOM 94 0 GLNA 19 -10.072 32.083 37.6951.00 37.10 A
ATOM 95 N LEUA 20 -10.821 31.949 39.7901.00 31.93 A
ATOM 96 CA LEUA 20 -10.729 33.414 39.9281.00 30.19 A
ATOM 97 CB LEUA 20 -11.811 33.972 40.8641.00 31.42 A
ATOM 98 CG LEUA 20 -13.246 34.105 40.3391.00 35.07 A
ATOM 99 CD1 LEUA 20 -13.979 35.172 41.1791.00 34.87 A
ATOM 100 CD2 LEUA 20 -13.226 34.554 38.8911.00 34.43 A
ATOM 101 C LEUA 20 -9.383 33.893 40.4381.00 27.07 A
ATOM 102 0 LEUA 20 -8.738 34.721 39.8141.00 27.58 A
ATOM 103 N THRA 21 -8.964 33.383 41.5851.00 24.20 A
ATOM 104 CA THRA 21 -7.679 33.818 42.1541.00 23.31 A
ATOM 105 CB THRA 21 -7.886 34.596 43.4771.00 22.88 A
ATOM 106 OGl THRA 21 -8.645 33.787 44.3741.00 23.64 A
ATOM 107 CG2 THRA 21 -8.683 35.898 4 3.2321.00 22.54 A
ATOM 108 C THRA 21 -6.736 32.644 42.4421.00 23.22 A
ATOM 109 0 THRA 22 -5.842 32.757 43.2811.00 23.48 A
ATOM 110 N SERA 22 -6.948 31.512 41.7831.00 22.33 A
ATOM 111 CA SERA 22 -6.069 30.359 42.0111.00 22.54 A
ATOM 112 CB SERA 22 -6.518 29.553 43.2371.00 22.65 A
ATOM 113 OG SERA 22 -7.758 28,909 42.9981.00 24.68 A
ATOM 114 C SERA 22 -6.119 29.484 40.7731.00 22.86 A
ATOM 115 0 SERA 22 -6.958 29.678 39.8811.00 21.28 A
ATOM 116 N GLYA 23 -5.198 28.533 40.6891.00 21.88 A
ATOM 117 CA GLYA 23 -5.218 27.670 39.5301.00 21.62 A
ATOM 118 C GLYA 23 -4.258 26.524 39.7331.00 21.90 A
ATOM 119 0 GLYA 23 -3.533 26.484 40.7341.00 20.10 A
ATOM 120 N ALAA 24 -4.248 25.609 38.7701.00 22.43 A
ATOM 121 CA ALAA 24 -3.386 24.450 38.8321.00 21.38 A
ATOM 122 CB ALAA 24 -4.225 23.200 39.1291.00 21.35 A
ATOM 123 C ALAA 24 -2.702 24.353 37.4831.00 23.49 A
ATOM 124 0 ALAA 24 -3.282 24.702 36.4301.00 22.42 A
ATOM 125 N VALA 25 -1.438 23.939 37.5081.00 21.72 A
ATOM 126 CA VALA 25 -0.674 23.808 36.2811.00 23.76 A
ATOM 127 CB VALA 25 0.529 24.789 36.2331.00 26.05 A
CA 02373918 2002-02-28
63
ATOM 128 CG1 VALA 25 1.391 24.500 34.9781.00 26.10 A
ATOM 129 CG2 VALA 25 0.038 26.224 36.2091.00 31.11 A
ATOM 130 C VALA 25 -0.110 22.413 36.2381.00 24.23 A
ATOM 131 0 VALA 25 0.331 21.897 37.2741.00 23.93 A
ATOM 132 N TRPA 26 -0.115 21.798 35.0631.00 24.25 A
ATOM 133 CA TRPA 26 0.458 20.459 34.9161.00 24.49 A
ATOM 134 CB TRPA 26 -0.590 19.439 34.4951.00 28.72 A
ATOM 135 CG TRPA 26 -0.006 18.132 33.9341.00 32.20 A
ATOM 136 CD2 TRPA 26 -0.077 17.668 32.5671.00 33.87 A
ATOM 137 CE2 TRPA 26 0.516 16.374 32.5241.00 35.35 A
ATOM 138 CE3 TRPA 26 -0.592 18.220 31.3711.00 34.27 A
ATOM 139 CD1 TRPA 26 0.622 17.133 34.6421.00 33.50 A
ATOM 140 NE1 TRPA 26 0.935 16.070 33.8011.00 35.95 A
ATOM 141 CZ2 TRPA 26 0.605 15.621 31.3421.00 35.87 A
ATOM 142 CZ3 TRPA 26 -0.504 17.472 30.1911.00 34.20 A
ATOM 143 CH2 TRPA 26 0.090 16.182 30.1891.00 35:90 A
ATOM 144 C TRPA 26 1.509 20.527 33.8291.00 25.41 A
ATOM 145 0 TRPA 26 1.363 21.274 32.8281.00 23.08 A
ATOM 146 N VALA 27 2.575 19.746 34.0001.00 22.86 A
ATOM 147 CA VALA 27 3.626 19.745 33.0021.00 23.49 A
ATOM 148 CB VALA 27 4.733 20.742 33.3421.00 25.20 A
ATOM 149 CG1 VALA 27 5.803 20.704 32.2371.00 25.19 A
ATOM 150 CG2 VALA 27 4.136 22.176 33.4301.00 25.58 A
ATOM 151 C VALA 27 4.221 18.361 32.9151.00 24.58 A
ATOM 152 O VALA 27 4.435 17.713 33.9431.00 21.32 A
ATOM 153 N GLNA 28 4.421 17.891 31.6881.00 23.14 A
ATOM 154 CA GLNA 28 5.029 16.590 31.4901.00 27.34 A
ATOM 155 CB GLNA 28 4.051 15.642 30.8121.00 30.52 A
ATOM 156 CG GLNA 28 4.662 14.304 30.5391.00 37.65 A
ATOM 157 CD GLNA 28 3.611 13.255 30.2311.00 41.99 A
ATOM 158 OE1 GLNA 28 2.730 13.465 29.3781.00 43.60 A
ATOM 159 NE2 GLNA 28 3.696 12.110 30.9241.00 42.95 A
ATOM 160 C GLNA 28 6.282 16.778 30.6401.00 26.40 A
ATOM 161 0 GLNA 28 6.239 17.410 29.5761.00 23.98 A
ATOM 162 N PHEA 29 7.403 16.247 31.1221.00 24.75 A
ATOM 163 CA PHEA 29 8.658 16.395 30.4231.00 24.70 A
ATOM 164 CB PHEA 29 9.781 16.608 31.4231.00 26.58 A
ATOM 165 CG PHEA 29 9.580 17.805 32.2951.00 25.63 A
ATOM 166 CD1 PHEA 29 9.006 17.669 33.5631.00 26.22 A
ATOM 167 CD2 PHEA 29 9.966 19.071 31.8511.00 25.52 A
ATOM 168 CE1 PHEA 29 8.815 18.782 34.3801.00 24.43 A
ATOM 169 CE2 PHEA 29 9.786 20.184 32.6451.00 25.07 A
ATOM 170 CZ PHEA 29 9.207 20.045 33.9231.00 26.84 A
ATOM 171 C PHEA 29 8.996 15.236 29.5061.00 25.07 A
ATOM 172 0 PHEA 29 8.348 14.185 29.5591.00 24.61 A
ATOM 173 N ASNA 30 10.027 15.400 28.6851.00 25.40 A
ATOM 174 CA ASNA 30 10.347 14.329 27.7421.00 28.37 A
ATOM 175 CB ASNA 30 11.397 14.786 26.7271.00 28.60 A
ATOM 176 CG ASNA 30 12.721 15.053 27.3631.00 33.92 A
ATOM 177 OD1 ASNA 30 12.813 15.827 28.3201.00 34.59 A
ATOM 178 ND2 ASNA 30 13.780 14.407 26.8441.00 36.62 A
ATOM 179 C ASNA 30 10.812 13.048 28.4101.00 27.80 A
ATOM 180 0 ASNA 30 10.751 11.990 27.7901.00 28.37 A
ATOM 181 N ASPA 31 11.267 13.134 29.6621.00 26.69 A
ATOM 182 CA ASPA 31 11.741 11.951 30.3711.00 27.04 A
ATOM 183 CB ASPA 31 12.777 12.327 31.4321.00 27.21 A
ATOM 184 CG ASPA 31 12.207 13.219 32.5281.00 27.15 A
ATOM 185 OD1 ASPA 31 11.000 13.506 32.5341.00 25.45 A
ATOM 186 OD2 ASPA 31 12.979 13.628 33.4011.00 27.89 A
ATOM 187 C ASPA 31 10.612 11.178 31.0201.00 27.64 A
ATOM 188 O ASPA 31 10.855 10.187 31.7001.00 25.26 A
ATOM 189 N GLYA 32 9.375 11.613 30.7861.00 26.83 A
CA 02373918 2002-02-28
64
ATOM 190 CA GLYA 32 8.242 10.921 31.3761.00 25.75 A
ATOM 191 C GLYA 32 7.840 11.475 32.7341.00 25.58 A
ATOM 192 0 GLYA 32 6.804 11.089 33.2731.00 26.85 A
ATOM 193 N SERA 33 8.631 12.373 33.3071.00 25.39 A
ATOM 194 CA SERA 33 8.271 12.878 34.6321.00 24.37 A
ATOM 195 CB SERA 33 9.485 13.468 35.3561.00 23.52 A
ATOM 196 OG SERA 33 10.048 14.588 34.6761.00 21.76 A
ATOM 197 C SERA 33 7.177 13.923 34.5011.00 24.25 A
ATOM 198 O SERA 33 6.883 14.385 33.3861.00 22.02 A
ATOM 199 N GLNA 34 6.565 14.264 35.6281.00 22.89 A
ATOM 200 CA GLNA 34 5.475 15.245 35.6481.00 24.56 A
ATOM 201 CB GLNA 34 4.111 14.551 35.5841.00 25.19 A
ATOM 202 CG GLNA 34 3.920 13.489 34.5371.00 30.40 A
ATOM 203 CD GLNA 34 2.618 12.744 34.7641.00 33.54 A
ATOM 204 OE1 GLNA 34 1.532 13.323 34.6291.00 32.79 A
ATOM 205 NE2 GLNA 34 2.713 11.464 35.1431.00 32.46 A
ATOM 206 C GLNA 34 5.455 16.073 36.9271.00 24.21 A
ATOM 207 O GLNA 34 5.776 15.580 38.0151.00 23.24 A
ATOM 208 N LEUA 35 5.025 17.324 36.7831.00 22.82 A
ATOM 209 CA LEUA 35 4.867 18.239 37.9061.00 21.35 A
ATOM 210 CB LEUA 35 5.722 19.501 37.7281.00 20.77 A
ATOM 211 CG LEUA 35 7:243 19.483 37.9111.00 T9.81 A
ATOM 212 CD1 LEUA 35 7.865 20.779 37.3811.00 19.63 A
ATOM 213 CD2 LEUA 35 7.529 19.316 39.4021.00 19.87 A
ATOM 214 C LEUA 35 3.393 18.676 37.8671.00 22.42 A
ATOM 215 0 LEUA 35 2.844 18.900 36.7801.00 19:09 A
ATOM 216 N VALA 36 2.752 18.744 39.0301.00 21.77 A
ATOM 217 CA VALA 36 1.376 19.250 39.1311.00 23.89 A
ATOM 218 CB vALA 36 0.344 18.161 39.5581.00 26.38 A
ATOM 219 CG1 vALA 36 -1.021 18.822 39.8581.00 25.49 A
ATOM 220 CG2 VALA 36 0.152 17.146 38.4341.00 22.63 A
ATOM 221 C VALA 36 1.553 20.285 40.2291.00 26.68 A
ATOM 222 O VALA 36 2.053 19.964 41.3241.00 25.04 A
ATOM 223 N META 37 1.178 21.532 39.9331.00 25.65 A
ATOM 224 CA META 37 1.352 22.615 40.8781.00 26.2 6 A
ATOM 225 CB META 37 2.440 23.554 40.3561.00 25.44 A
ATOM 226 CG META 37 3.613 22.779 39.7561.00 30.14 A
ATOM 227 SD META 37 4.975 23.772 39.1961.00 32.21 A
ATOM 228 CE META 37 4.108 24.902 38.1871.00 30.46 A
ATOM 229 C META 37 0.080 23.398 41.0901.00 25.73 A
ATOM 230 0 META 37 -0.753 23.513 40.1701.00 27.70 A
ATOM 231 N GLNA 38 -0.115 23.903 42.3041.00 23.37 A
ATOM 232 CA GLNA 38 -1.292 24.728 42.5451.00 22.97 A
ATOM 233 CB GLNA 38 -2.157 24.137 43.6441.00 24.17 A
ATOM 234 CG GLNA 38 -2.892 22.891 43.1161.00 26.79 A
ATOM 235 CD GLNA 38 -3.932 22.394 44.0541.00 28.97 A
ATOM 236 OE1 GLNA 38 -4.754 23.164 44.5371.00 31.62 A
ATOM 237 NE2 GLNA 38 -3.930 21.093 44.3141.00 31.46 A
ATOM 238 C GLNA 38 -0.711 26.095 42.8901.00 22.53 A
ATOM 239 O GLNA 38 0.348 26.187 43.5301.00 21.65 A
ATOM 240 N ALAA 39 -1.365 27.153 42.4101.00 21.23 A
ATOM 241 CA ALAA 39 -0.882 28.517 42.6151.00 20.50 A
ATOM 242 CB ALAA 39 -0.262 29.028 41.3051.00 20.80 A
ATOM 243 C ALAA 39 -2.023 29.450 43.0561.00 21.59 A
ATOM 244 O ALAA 39 -3.178 29.095 42.9271.00 19.34 A
ATOM 245 N GLYA 40 -1.692 30.645 43.5421.00 21.15 A
ATOM 246 CA GLYA 40 -2.733 31.570 43.9941.00 23.40 A
ATOM 247 C GLYA 40 -2.278 33.015 43.8711.00 22.86 A
ATOM 248 0 GLYA 40 -1.081 33.294 43.8731.00 22.47 A
ATOM 249 N VALA 41 -3.230 33.939 43.7641.00 22.89 A
ATOM 250 CA VALA 41 -2.913 35.355 43.6281.00 22.01 A
ATOM 251 CB VALA 41 -4.080 36.073 42.8881.00 22.10 A
CA 02373918 2002-02-28
ATOM 252 CG1 VALA 41 -3.840 37.57742.814 1.00 22.14 A
ATOM 253 CG2 VALA 41 -4.228 35.47841.469 1.00 21.24 A
ATOM 254 C VALA 41 -2.728 35.94845.039 1:00 2-1.72 A
ATOM 255 0 VALA 41 -3.617 35.79145.900 1.00 21.08 A
5 ATOM 256 N SERA 42 -1.599 36.62445.287 1.00 18.13 A
ATOM 257 CA SERA 42 -1.355 37.23846.608 1.00 19.79 A
ATOM 258 CB SERA 42 0.132 37.20546.980 1.00 19.38 A
ATOM 259 OG SERA 42 0.925 37.57045.856 1.00 18.21 A
ATOM 260 C SERA 42 -1.786 38.69646.642 1.00 2'1.07 A
10 ATOM 261 0 SERA 42 -1.907 39.28347.721 1.00 20.90 A
ATOM 262 N SERA 43 -1.931 39.31045.474 1.00 20.00 A
ATOM 263 CA SERA 43 -2.395 40.70545.442 1.00 20.47 A
ATOM 264 CB SERA 43 -1.286 41.68545.872 1.00 22.34 A
ATOM 265 OG SERA 43 -0.244 41.75844.915 1.00 27.90 A
15 ATOM 266 C SERA 43 -2.918 41.08944.079 1.00 20.54 A
ATOM 267 0 SERA 43 -2.405 40.64343.031 1.00 18.49 A
ATOM 268 N ILEA 44 -3.965 41.90744.099 1.00 19.49 A
ATOM 269 CA ILEA 44 -4.591 42.38542.891 1.00 20.54 A
ATOM 270 CB ILEA 44 -6.049 41.89342.793 1.00 22.31 A
20 ATOM 271 CG2 ILEA 44 -6.695 42.44441.543 1.00 19.72 A
ATOM 272 CG1 ILEA 44 -6.084 40.35142.790 1.00 22.18 A
ATOM 273 CD1 ILEA 44 -7.485 39.72442.708 1.00 23.31 A
ATOM 274 C ILEA 44 -4.577 43.90942.905 1.00 22.19 A
ATOM 275 0 ILEA 44 -5.088 44.54643.843 1.00 20.82 A
25 ATOM 276 N SERA 45 -3.969 44.48741.881 1.00 21.03 A
ATOM 277 CA SERA 45 -3.901 45.93941.747 1.00 22.60 A
ATOM 278 CB SERA 45 -2.449 46.37241.535 1.00 2b.70 A
ATOM 279 OG SERA 45 -2.321 47.78241.474 1.00 27.90 A
ATOM 280 C SERA 45 -4.756 46.26240.531 1.00 22.78 A
30 ATOM 281 0 SERA 45 -4.403 45.92839.377 1.00 22.24 A
ATOM 282 N TYRA 46 -5.901 46.88040.798 1.00 22.65 A
ATOM 283 CA TYRA 46 -6.862 47.22539.763 1.00 22.55 A
ATOM 284 CB TYRA 46 -8.269 46.83140.218 1.00 22.32 A
ATOM 285 CG TYRA 46 -9.361 47.21939.231 1.00 25.37 A
35 ATOM 286 CD1 TYRA 46 -9.518 46.50838.037 1.00 24.33 A
ATOM 287 CE1 TYRA 46 -10.515 46.85037.108 1.00 24.37 A
ATOM 288 CD2 TYRA 46 -10.246 48.30339.488 1.00 24.12 A
ATOM 289 CE2 TYRA 46 -11.254 48.65238.562 1.00 23.53 A
ATOM 290 CZ TYRA 46 -11.375 47.92037.373 1.00 25.93 A
40 ATOM 291 OH TYRA 46 -12.325 48.23336.425 1.00 24.28 A
ATOM 292 C TYRA 46 -6.842 48.72039.455 1.00 24.28 A
ATOM 293 0 TYRA 46 -6.968 49.56540.357 1.00 23.43 A
ATOM 294 N THRA 47 -6.656 49.04138.183 1.00 23.62 A
ATOM 295 CA THRA 47 -6.675 50.42137.739 1.00 24.08 A
45 ATOM 296 CB THRA 47 -5.469 50.73236.843 1.00 24.70 A
ATOM 297 OG1 THRA 47 -4.278 50.54937.615 1.00 25.79 A
ATOM 298 CG2 THRA 47 -5.504 52.20236.344 1.00 24.86 A
ATOM 299 C THRA 47 -7.970 50.59736.953 1.00 23.37 A
ATOM 300 0 THRA 47 -8.169 49.95735.926 1.00 22.53 A
50 ATOM 301 N SERA 48 -8.863 51.42737.478 1.00 22.12 A
ATOM 302 CA SERA 48 -10.145 51.70936.838 1.00 20.42 A
ATOM 303 CB SERA 48 -11.014 52.57737.771 1.00 22.04 A
ATOM 304 OG SERA 48 -12.030 53.28537.022 1.00 23.62 A
ATOM 305 C SERA 48 -9.967 52.45935.543 1.00 19.42 A
55 ATOM 306 0 SERA 48 -8.908 53.04035.279 1,00 19.34 A
ATOM 307 N PROA 49 -11.002 52.45434.691 1.00 19.82 A
ATOM 308 CD PROA 49 -12.265 51.69334.726 1.00 20.00 A
ATOM 309 CA PROA 49 -10.871 53.19333.442 1.00 21.05 A
ATOM 310 CB PROA 49 -12.221 52.98032.778 1.00 21.03 A
60 ATOM 311 CG PROA 49 -12.626 51.62633.274 1:00 22.27 A
ATOM. 312 C PROA 49 -10.644 54:67633.777 1.00 23.30 A
ATOM 313 0 PROA 49 -10.110 55.41632.958 1.00 24.51 A
CA 02373918 2002-02-28
66
ATOM 314 N ASPA 50 -11.065 55.118 34.9721.00 22.65 A
ATOM 315 CA ASPA 50 -10.882 56.531 35.3381.00 24.70 A
ATOM 316 CB ASPA 50 -11.983 57.025 36.3121.00 21.88 A
ATOM 317 CG ASPA 50 -11.898 56.421 37.7071.00 24.64 A
ATOM 318 OD1 ASPA 50 -10.847 55.844 38.0521.00 22.86 A
ATOM 319 OD2 ASPA 50 -12.899 56.547 38.4851.00 22.31 A
ATOM 320 C ASPA 50 -9.491 56.860 35.8761.00 23.99 A
ATOM 321 O ASPA 50 -9.240 57.980 36.3121.00 23.78 A
ATOM 322 N GLYA 51 -8.579 55.887 35.8331.00 23.93 A
ATOM 323 CA GLYA 51 -7.207 56.127 36.2941.00 22.64 A
ATOM 324 C GLYA 51 -6.904 55.899 37.7621.00 23:25 A
ATOM 325 0 GLYA 51 -5.742 55.965 38.1611.00 2'5.45 A
ATOM 326 N GLNA 52 -7.918 55.629 38:5801.00 22.74 A
ATOM 327 CA GLNA 52 -7.688 55.398 40.0061.00 24.31 A
ATOM 328 CB GLNA 52 -8.971 55.644 40.8051.00 25.55 A
ATOM 329 CG GLNA 52 -9.422 57.116 40.8401.00 27.26 A
ATOM 330 CD GLNA 52 -8.415 57.965 41.5811.00 28.20 A
ATOM 331 OE1 GLNA 52 -7.610 58.667 40.9761.00 28.60 A
ATOM 332 NE2 GLNA 52 -8.439 57.879 42.9051.00 30.60 A
ATOM 333 C GLNA 52 -7.235 53.951 40.2411.00 23.71 A
ATOM 334 O GLNA 52 -7.838 53.023 39.7091.00 22.37 A
ATOM 335 N THRA 53 -6.206 53.762 41.0531.00 23.96 A
ATOM 336 CA THRA 53 -5.730 52.404 41.3281.00 26.22 A
ATOM 337 CB THRA 53 -4.203 52.267 41.0931.00 26.01 A
ATOM 338 OG1 THRA 53 -3.922 52.437 39.7011.00 27.10 A
ATOM 339 CG2 THRA 53 -3.721 50.858 41.5001.00 27.86 A
ATOM 340 C THRA 53 -6.036 51.967 42.7461.00 27.22 A
ATOM 341 0 THRA 53 -5.855 52.743 43.6891.00 28.02 A
ATOM 342 N THRA 54 -6.513 50.726 42.8881.00 24.96 A
ATOM 343 CA THRA 54 -6.840 50.159 44.1901.00 25.21 A
ATOM 344 CB THRA 54 -8:368 50.007 44.3901.00 27.26 A
ATOM 345 OG1 THRA 54 -9.035 51.243 44.0701.00 29.29 A
ATOM 346 CG2 THRA 54 -8.658 49.649 45.8301.00 28.65 A
ATOM 347 C THRA 54 -6.200 48.771 44.3041.00 24.23 A
ATOM 348 0 THRA 54 -6.286 47.951 43.3811.00 21.76 A
ATOM 349 N ARGA 55 -5.523 48.524 45.4191.00 23.84 A
ATOM 350 CA ARGA 55 -4.881 47.236 45.6341.00 23.81 A
ATOM 351 CB ARGA 55 -3.453 47.456 46.1461.00 24.59 A
ATOM 352 CG ARGA 55 -2.679 46.172 46.4501.00 32.74 A
ATOM 353 CD ARGA 55 -1.368 46.412 47.2411.00 34.85 A
ATOM 354 NE ARGA 55 -0.955 45.153 47.8631.00 42.09 A
ATOM 355 CZ ARGA 55 -1.475 44.645 48.9831.00 42.52 A
ATOM 356 NH1 ARGA 55 -2.429 45.293 49.6491.00 44.55 A
ATOM 357 NH2 ARGA 55 -1:072 43.454 49.4141.00 42.98 A
ATOM 358 C ARGA 55 -5.684 46.398 46.6381.00 25.42 A
ATOM 359 O ARGA 55 -6.163 46.906 47.6761.00 24.29 A
ATOM 360 N TYRA 56 -5.858 45.118 46.3221.00 22.94 A
ATOM 361 CA TYRA 56 -6.556 44.210 47.2271.00 24.10 A
ATOM 362 CB TYRA 56 -7.803 43.627 46.5631.00 24.74 A
ATOM 363 CG TYRA 56 -8.775 44.697 46.1151.00 25.46 A
ATOM 364 CD1 TYRA 56 -8.568 45.387 44.9181.00 25.25 A
ATOM 365 CE1 TYRA 56 -9.432 46.391 44.5041.00 27.98 A
ATOM 366 CD2 TYRA 56 -9.884 45.041 46.9001.00 26.40 A
ATOM 367 CE2 TYRA 56 -10.765 46.054 4 6.4961.00 28.27 A
ATOM 368 CZ TYRA 56 -10.526 46.720 45.2941.00 29.75 A
ATOM 369 OH TYRA 56 -11.372 47.718 44.8761.00 33.43 A
ATOM 370 C TYRA 56 -5.613 43.085 47.6381.00 23.96 A
ATOM 371 0 TYRA 56 -5.070 42.367 46.7891.00 23.65 A
ATOM 372 N GLYA 57 -5.377 42.979 48.9361.00 22.44 A
ATOM 373 CA GLYA 57 -4.519 41.939 49.4501.00 25.69 A
ATOM 374 C GLYA 57 -5.270 40.617 49.4041.00 25.67 A
ATOM 375 0 GLYA 57 -6.484 40.589 49.1741.00 23.94 A
CA 02373918 2002-02-28
67
ATOM 376 N GLUA 58 -4.558 39.523 49.6591.00 26.09 A
ATOM 377 CA GLUA 58 -5.161 38.205 49.5801.00 26.78 A
ATOM 378 CB GLUA 58 -4.079 37.145 49.7981.00 28.97 A
ATOM 379 CG GLUA 58 -4.507 35.750 49.4031.00 31.47 A
ATOM 380 CD GLUA 58 -3.325 34.781 49.1861.00 34.27 A
ATOM 381 OE1 GLUA 58 -3.614 33.595 48.9431.00 35.62 A
ATOM 382 OE2 GLUA 58 -2.129 35.193 49.2431.00 32.05 A
ATOM 383 C GLUA 58 -6:305 38.002 50.5571.00 27.07 A
ATOM 384 0 GLUA 58 -7.247 37.245 50.2721.00 26.31 A
ATOM 385 N ASNA 59 -6.222 38.666 51.7101.00 25.53 A
ATOM 386 CA ASNA 59 -7.260 38.535 52.7261.00 27.19 A
ATOM 387 CB ASNA 59 -6.609 38.444 54.1081.00 26.47 A
ATOM 388 CG ASNA 59 -5.726 37.212 54.2341.00 28.80 A
ATOM 389 OD1 ASNA 59 -5.957 36.209 53.5371.00 28.28 A
ATOM 390 ND2 ASNA 59 -4.736 37.261 55.1131.00 27.05 A
ATOM 391 C ASNA 59 -8.351 39.631 52.7141.00 28.83 A
ATOM 392 0 ASNA 59 -9.052 39.823 53.6971.00 3'0.01 A
ATOM 393 N GLUA 60 -8.507 40.326 51.5971.00 30.40 A
ATOM 394 CA GLUA 60 -9.534 41.348 51.5071.00 31.99 A
ATOM 395 CB GLUA 60 -8.966 42.637 50.9451.00 30.74 A
ATOM 396 CG GLUA 60 -7:958 43.296 51.8441.00 33.45 A
ATOM 397 CD GLUA 60 -7.447 44.568 51.2201.00 34.00 A
ATOM 398 OE1 GLUA 60 -8.260 45.494 51.0271.00 37.35 A
ATOM 399 OE2 GLUA 60 -6.248 44.643 50.9151.00 32.39 A
ATOM 400 C GLUA 60 -10.625 40.843 50:5951.00 32.78 A
ATOM 401 O GLUA 60 -10.363 40.126 49.6201.00 33.16 A
ATOM 402 N LYSA 61 -11.861 41.206 50.9071.00 33.50 A
ATOM 403 CA LYSA 61 -12.979 40.784 50.0801.00 34.01 A
ATOM 404 CB LYSA 61 -14.288 41.055 50.8181.00 36.55 A
ATOM 405 CG LYSA 61 -15.504 40.449 50.1411.00 41.45 A
ATOM 406 CD LYSA 61 -16.787 40.806 50.8921.00 44.29 A
ATOM 407 CE LYSA 61 -18.013 40.305 50.1531.00 44.74 A
ATOM 408 NZ LYSA 61 -19.239 40.572 50.9651.00 47.69 A
ATOM 409 C LYSA 61 -12.906 41.578 48.7751.00 33.44 A
ATOM 410 0 LYSA 61 -12.568 42.769 48.7841.00 33.86 A
ATOM 411 N LEUA 62 -13.187 40.927 47.6541.00 32.05 A
ATOM 412 CA LEUA 62 -13.142 41.607 46.3711.00 33.14 A
ATOM 413 CB LEUA 62 -12.623 40.678 45.2581.00 32.95 A
ATOM 414 CG LEUA 62 -11.187 40.165 45.4211.00 35.06 A
ATOM 415 CD1 LEUA 62 -10.804 39.335 44.1931.00 35.29 A
ATOM 416 CD2 LEUA 62 -10.228 41.344 45.6061.00 34.89 A
ATOM 417 C LEUA 62 -14.526 42.093 4 5.9771.00 33.44 A
ATOM 418 O LEUA 62 -15.513 41.366 46.1151.00 34.29 A
ATOM 419 N PROA 63 -14.626 43.342 45:5061.00 33.07 A
ATOM 420 CD PROA 63 -13.582 44.368 45.3471.00 32.27 A
ATOM 421 CA PROA 63 -15.944 43:839 45.1031.00 32.15 A
ATOM 422 CB PROA 63 -15.681 45.309 44.7611.00 32.76 A
ATOM 423 CG PROA 63 -14.223 45:326 44.3571.00 32.85 A
ATOM 424 C PROA 63 -16.410 43.017 43.9021.00 32.47 A
ATOM 425 O PROA 63 -15.588 42.416 43.1771.00 31.14 A
ATOM 426 N GLUA 64 -17.721 42.968 43.6851.00 31.52 A
ATOM 427 CA GLUA 64 -18.254 42.189 42.5691.00 32.30 A
ATOM 428 CB GLUA 64 -19.790 42.237 42.5361.00 36.47 A
ATOM 429 CG GLUA 64 -20.457 41.249 43.4751.00 41.60 A
ATOM 430 CD GLUA 64 -19.936 39.825 43.2891.00 44.51 A
ATOM 431 OE1 GLUA 64 -20.046 39.283 42.1621.00 46.75 A
ATOM 432 OE2 GLUA 64 -19.417 39.258 44.2791.00 45.99 A
ATOM 433 C GLUA 64 -17.752 42.548 41.1851.00 29.49 A
ATOM 434 0 GLUA 64 -17.537 41.660 40.3591.00 26.90 A
ATOM 435 N TYRA 65 -17.577 43.836 40.9051.00 27.49 A
ATOM 436 CA TYRA 65 -17.138 44.200 39.5651.00 27.12 A
ATOM 437 CB TYRA 65 -17.216 45.715 39.3681.00 27.10 A
CA 02373918 2002-02-28
68
ATOM 438 CG TYRA 65 -16.285 46.550 40.2141.00 27.17 A
ATOM 439 CD1 TYRA 65 -14.975 46.816 39.8031.00 25.51 A
ATOM 440 CE1 TYRA 65 -14.143 47.621 40.5581.00 27.31 A
ATOM 441 CD2 TYRA 65 -16.731 47.109 41.4111.00 26.81 A
ATOM 442 CE2 TYRA 65 -15.911 47.904 42.1741.00 28.77 A
ATOM 443 CZ TYRA 65 -14.627 48.160 41.7451.00 29.82 A
ATOM 444 OH TYRA 65 -13.844 48.979 42.5081.00 33.48 A
ATOM 445 C TYRA 65 -15.736 43.661 39.2201.00 24.82 A
ATOM 446 O TYRA 65 -15.431 43.405 38.0441.00 23.19 A
ATOM 447 N ILEA 66 -14.892 43:481 40.2351.00 25.20 A
ATOM 448 CA ILEA 66 -13.556 42.922 40.0071.00 26.39 A
ATOM 449 CB ILEA 66 -12.598 43.255 41.1811.00 26.58 A
ATOM 450 CG2 ILEA 66 -11.315 42.405 41.0871.00 27.43 A
ATOM 451 CG1 ILEA 66 -12.227 44.749 41.1141.00 29.08 A
ATOM 452 CD1 ILEA 66 -11.185 45.175 42.1121.00 30.16 A
ATOM 453 C ILEA 66 -13.678 41.397 39.8101.00 26.68 A
ATOM 454 0 ILEA 66 -13.010 40.823 38.9561.00 27.11 A
ATOM 455 N LYSA 67 -14.533 40.745 40.5911.00 27.91 A
ATOM 456 CA LYSA 67 -14.740 39.306 40.4371.00 29.38 A
ATOM 457 CB LYSA 67 -15.749 38.786 4 1.4651.00 30.38 A
ATOM 458 CG LYSA 67 -15:295 38.884 42.9021.00 32.38 A
ATOM 459 CD LYSA 67 -16.330 38.263 43.8451.00 36.43 A
ATOM 460 CE LYSA 67 -15.836 38.315 45.2891.00 40.14 A
ATOM 461 NZ LYSA 67 -16.840 37.774 46.2671.00 44.58 A
ATOM 462 C LYSA 67 -15.252 38.987 39.0191.00 29.85 A
ATOM 463 0 LYSA 67 -14.776 38.052 38.3831.00 28.01 A
ATOM 464 N GLNA 68 -16.207 39.775 38.5151.00 30.12 A
ATOM 465 CA GLNA 68 -16.756 39.546 37.1801.00 2'9.47 A
ATOM 466 CB GLNA 68 -17.900 40.526 36.8831.00 33.01 A
ATOM 467 CG GLNA 68 -18.977 40.570 37.9441.00 35.71 A
ATOM 468 CD GLNA 68 -20.199 41.372 37.4981.00 39.88 A
ATOM 469 OE1 GLNA 68 -20.089 42.332 36.7171.00 42.71 A
ATOM 470 NE2 GLNA 68 -21.368 40.990 38.0021.00 40.73 A
ATOM 471 C GLNA 68 -15.686 39.691 36.1031.00 29.74 A
ATOM 472 O GLNA 68 -15.732 38.991 35.0811.00 3'0.02 A
ATOM 473 N LYSA 69 -14.734 40.611 36.2971.00 26.78 A
ATOM 474 CA LYSA 69 -13.669 40.769 35.3111.00 26.10 A
ATOM 475 CB LYSA 69 -12.981 42.137 35.4391.00 23.05 A
ATOM 476 CG LYSA 69 -13.695 43.222 34.5921.00 21.87 A
ATOM 477 CD LYSA 69 -13:365 44.666 35.0371.00 19.54 A
ATOM 478 CE LYSA 69 -14.012 45.686 34.0811.00 20.65 A
ATOM 479 NZ LYSA 69 -13.682 47.106 34.4331.00 2'0.86 A
ATOM 480 C LYSA 69 -12.658 39.619 35.4501.00 24.70 A
ATOM 481 0 LYSA 69 -12.119 39.146 34.4471.00 24.05 A
ATOM 482 N LEUA 70 -12.420 39.162 36.6801.00 26.08 A
ATOM 483 CA LEUA 70 -11.507 38.025 36.9141.00 27.98 A
ATOM 484 CB LEUA 70 -11.383 37.719 38.4111.00 27.23 A
ATOM 485 CG LEUA 70 -10.489 38.642 39.2281.00 28.37 A
ATOM 486 CD1 LEUA 70 -10.655 38:372 40.7201.00 28.59 A
ATOM 487 CD2 LEUA 70 -9.052 38.415 38.7731.00 26.77 A
ATOM 488 C LEUA 70 -12.060 36.778 36.2301.00 29.99 A
ATOM 489 O LEUA 70 -11.314 35.981 35.6661.00 31.87 A
ATOM 490 N GLNA 71 -13.374 36.602 36.2961.00 31.27 A
ATOM 491 CA GLNA 71 -13.995 35.440 35.6841.00 33.89 A
ATOM 492 CB GLNA 71 -15:491 35.414 35.9851.00 36.26 A
ATOM 493 CG GLNA 71 -16.246 34.336 35.2121.00 42.00 A
ATOM 494 CD GLNA 71 -15:826 32.922 35.6001.00 45.50 A
ATOM 495 OE1 GLNA 71 -15.847 32.567 36.7861.00 47.23 A
ATOM 496 NE2 GLNA 71 -15.447 32.104 34.6031.00 45.34 A
ATOM 497 C GLNA 71 -13.777 35.386 34.1811.00 34.10 A
ATOM 498 0 GLNA 71 -13.872 34.319 33.5811.00 33.80 A
ATOM 499 N LEUA 72 -13.486 36.526 33.5611.00 32.87 A
CA 02373918 2002-02-28
69
ATOM 500 CA LEUA 72 -13.271 36.53332.119 1.00 32.81 A
ATOM 501 CB LEUA 72 -13.268 37.97431:574 1,00 30.82 A
ATOM 502 CG LEUA 72 -14.599 38.75931.635 1.00 30.29 A
ATOM 503 CD1 LEUA 72 -14.395 40.20631.148 1.00 27.81 A
ATOM 504 CD2 LEUA 72 -15.638 38.04730.772 1.00 30.00 A
ATOM 505 C LEUA 72 -11.933 35.85831.824 1.00 33.11 A
ATOM 506 O LEUA 72 -11.645 35.49030.684 1.00 31.50 A
ATOM 507 N LEUA 73 -11.124 35.68732.866 1.00 32.72 A
ATOM 508 CA LEUA 73 -9.806 35.06532.714 1.00 33.81 A
ATOM 509 CB LEUA 73 -8:796 35.75433.644 1.00 3;5.28 A
ATOM 510 CG LEUA 73 -8.559 37.21833.265 1.00 37.42 A
ATOM 511 CD1 LEUA 73 -7.923 37.97534.426 1.00 37.87 A
ATOM 512 CD2 LEUA 73 -7.678 37.25132.017 1.00 37.72 A
ATOM 513 C LEUA 73 -9.781 33.55832.963 1.00 32.63 A
ATOM 514 O LEUA 73 -8.872 32.85932.496 1.00 33.32 A
ATOM 515 N SERA 74 -10.774 33.04733.676 1.00 30.99 A
ATOM 516 CA SERA 74 -10.802 31.61733.980 1.00 2'9.90 A
ATOM 517 CB SERA 74 -12.037 31.28634.803 1.00 30.28 A
ATOM 518 OG SERA 74 -12.151 32.19735.893 1.00 37.30 A
ATOM 519 C SERA 74 -10.782 30.75132.719 1.00 29.33 A
ATOM 520 0 SERA 74 -11.611 30.92831.811 1.00 27.15 A
ATOM 521 N SERA 75 -9.868 29.78132.676 1.00 27.21 A
ATOM 522 CA SERA 75 -9:778 28.92031.506 1.00 25.21 A
ATOM 523 CB SERA 75 -9.041 29.63830.379 1.00 26.98 A
ATOM 524 OG SERA 75 -7.649 29.73630.653 1.00 27.61 A
ATOM 525 C SERA 75 -9.072 27.60431.773 1.00 25.16 A
ATOM 526 O SERA 75 -8.431 27.43432.812 1.00 21.93 A
ATOM 527 N ILEA 76 -9.213 26.68630.816 1.00 24.41 A
ATOM 528 CA ILEA 76 -8.588 25.35830.855 1.00 24.95 A
ATOM 529 CB ILEA 76 -9.603 24.22230.750 1.00 26.86 A
ATOM 530 CG2 ILEA 76 -8.850 22.88530.655 1.00 27.48 A
ATOM 531 CG1 ILEA 76 -10.605 24.29531.888 1.00 29.55 A
ATOM 532 CD1 ILEA 76 -10.016 24.02033.204 1.00 33.33 A
ATOM 533 C ILEA 76 -7.789 25.28529.562 1.00 25.08 A
ATOM 534 O ILEA 76 -8.340 25.53428.481 1.00 24.07 A
ATOM 535 N LEUA 77 -6.511 24.93929.654 1.00 22.73 A
ATOM 536 CA LEUA 77 -5.707 24.83728.464 1.00 23.84 A
ATOM 537 CB LEUA 77 -4.752 26.02528.367 1.00 26.02 A
ATOM 538 CG LEUA 77 -3.734 25.93227.204 1.00 29.74 A
ATOM 539 CD1 LEUA 77 -3.488 27.32526.608 1.00 29.57 A
ATOM 540 CD2 LEUA 77 -2.447 25.31927.699 1.00 31.16 A
ATOM 541 C LEUA 77 -4.916 23.53228.513 1.00 23.98 A
ATOM 542 0 LEUA 77 -4.493 23.09229.581 1.00 23.14 A
ATOM 543 N LEUA 78 -4.769 22.89727.361 1.00 23.73 A
ATOM 544 CA LEUA 78 -3.982 21.67127.239 1.00 25.08 A
ATOM 545 CB LEUA 78 -4.906 20.46327:072 1.00 26.86 A
ATOM 546 CG LEUA 78 -5.700 19.99628.287 1.00 28.53 A
ATOM 547 CD1 LEUA 78 -6.688 18.90427.894 1.00 30:02 A
ATOM 548 CD2 LEUA 78 -4.715 19.45729.319 1.00 31.81 A
ATOM 549 C LEUA 78 -3.156 21.86025.973 1.00 25.44 A
ATOM 550 0 LEUA 78 -3.714 22.19424.930 1.00 25.36 A
ATOM 551 N META 79 -1:839 21.67426.055 1.00 24.42 A
ATOM 552 CA META 79 -0.973 21.80724.887 1.00 25.49 A
ATOM 553 CB META 79 -0.155 23.10024.950 1.00 27.60 A
ATOM 554 CG META 79 0.708 23.35323.706 1,00 33.94 A
ATOM 555 SD META 79 1.544 24.99523.654 1.00 38.48 A
ATOM 556 CE META 79 0.465 25.93724.741 1.00 36:27 A
ATOM 557 C META 79 -0.027 20.60524.841 1.00 26.1 6 A
ATOM 558 O META 79 0.612 20:28525.853 1.00 25.19 A
ATOM 559 N PHEA 80 0.061 19.94923.680 1.00 26.66 A
ATOM 560 CA PHEA 80 0.919 18.76723.497 1.00 28.28 A
ATOM 561 CB PHEA 80 0.076 17.50923.219 1.00 29:89 A
CA 02373918 2002-02-28
ATOM 562 CG PHEA 80 -1.082 17.32624.154 1.00 31.47 A
ATOM 563 CD1 PHEA 80 -2.205 18.15224.066 1.00 34.23 A
ATOM 564 CD2 PHEA 80 -1.036 16.35825.152 1.00 33.79 A
ATOM 565 CE1 PHEA 80 -3.269 18.01924.968 1.00 34.34 A
5 ATOM 566 CE2 PHEA 80 -2.098 16.21626.064 1.00 34.66 A
ATOM 567 CZ PHEA 80 -3.207 17.05025.967 1.00 34.41 A
ATOM 568 C PHEA 80 1.862 18.92122.309 1.00 29.99 A
ATOM 569 0 PHEA 80 1.463 19.44621.271 1.00 26.03 A
ATOM 570 N SERA 81 3.099 18.44422.447 1.00 31.47 A
10 ATOM 571 CA SERA 81 4.034 18.46521.325 1.00 35.92 A
ATOM 572 CB SERA 81 5.431 18.02221.764 1.00 36.10 A
ATOM 573 OG SERA 81 5.974 18.94522.705 1.00 40.41 A
ATOM 574 C SERA 81 3.453 17.42520.354 1.00 37.84 A
ATOM 575 O SERA 81 3.029 16.35020.778 1.00 38.67 A
15 ATOM 576 N ASNA 82 3.421 17.74319.063 1.00 39.87 A
ATOM 577 CA ASNA 82 2.859 16.83718.059 1.00 42.79 A
ATOM 578 CB ASNA 82 1.668 17.53017.374 1.00 44.12 A
ATOM 579 CG ASNA 82 0.881 16.60216.452 1.00 44.62 A
ATOM 580 OD1 ASNA 82 0.133 17.06515.584 1.00 46.36 A
20 ATOM 581 ND2 ASNA 82 1.032 15.29716.643 1.00 44.93 A
ATOM 582 C ASNA 82 3.930 16.46617.019 1.00 45.11 A
ATOM 583 0 ASNA 82 3.742 16.76015.809 1.00 45.06 A
ATOM 584 OXT ASNA 82 4.964 15.89217.439 1.00 48.72 A
ATOM 585 CB SERB 8 -20.703 44.76826.853 1.00 46.84 B
25 ATOM 586 OG SERB 8 -20.236 45.83126.037 1.00 49.95 B
ATOM 587 C SERB 8 -18.436 43.67126.952 1.00 44.17 B
ATOM 588 0 SERB 8 -17.598 43.93726.079 1.00 45.41 B
ATOM 589 N SERB 8 -20.548 42.34527.331 1.00 46.65 B
ATOM 590 CA SERB 8 -19.923 43.47526.579 1.00 45.64 B
30 ATOM 591 N VALB 9 -18.112 43.54828.239 1.00 40.04 B
ATOM 592 CA VALB 9 -16.731 43.71028.703 1.00 35.61 B
ATOM 593 CB VALB 9 -16.655 43.76130.262 1.00 35.13 B
ATOM 594 CG1 VALB 9 -15.189 43.76330.721 1.00 32.66 B
ATOM 595 CG2 VALB 9 -17.358 45.01830.785 1.00 33.73 B
35 ATOM 596 C VALB 9 -15.886 42.53428.230 1.00 33.45 B
ATOM 597 0 VALB 9 -16.322 41.40128.329 1.00 31:66 B
ATOM 598 N PHEB 10 -14.693 42.78727.695 1.00 31.66 B
ATOM 599 CA PHEB 10 -13.846 41.67427.283 1.00 31.75 B
ATOM 600 CB PHEB 10 -14.188 41.19925.846 1.00 33.57 B
40 ATOM 601 CG PHEB 10 -13.981 42.24224.765 1.00 38.37 B
ATOM 602 CD1 PHEB 10 -12.728 42.42324.180 1.00 39.12 B
ATOM 603 CD2 PHEB 10 -15.038 43.05324.342 1.00 39.71 B
ATOM 604 CE1 PHEB 10 -12.519 43.39723.192 1.00 39.97 B
ATOM 605 CE2 PHEB 10 -14.840 44.03623:352 1.00 40.15 B
45 ATOM 606 CZ PHEB 10 -13:578 44.20722.779 1.00 40.06 B
ATOM 607 C PHEB 10 -12.354 41.95827.429 1.00 29.89 B
ATOM 608 O PHEB 10 -11.918 43.12327.519 1.00 26.55 B
ATOM 609 N VALB 11 -11.576 40.87627.496 1.00 28.69 B
ATOM 610 CA VALB 11 -10.127 40.98527.617 1.00 27.06 B
50 ATOM 611 CB VALB 11 -9.458 39.61627.905 1.00 26.64 B
ATOM 612 CG1 VALB 11 -7.917 39.79327.932 1.00 25.73 B
ATOM 613 CG2 VALB 11 -9.940 39.06829.255 1.00 27.02 B
ATOM 614 C VALB 11 -9.587 41.49726.307 1.00 26.17 B
ATOM 615 0 VALB 11 -9.975 41.00625.249 1.00 27.06 B
55 ATOM 616 N LYSB 12 -8.692 42.47926.358 1.00 25.60 B
ATOM 617 CA LYSB 12 -8.135 43.00625.129 1.00 29.71 B
ATOM 618 CB LYSB 12 -8.219 44.52525.107 1.00 30.24 B
ATOM 619 CG LYSB 12 -8.134 45.08023.703 1.00 35.27 B
ATOM 620 CD LYSB 12 -7.894 46.57923.697 1.00 38.59 B
60 ATOM 621 CE LYSB 12 -7.611 47.05122.274 1.00 38:61 B
ATOM 622 NZ LYSB 12 -7.069 48.44122.283 1.00 43.07 B
ATOM 623 C LYSB 12 -6.682 42.58624.985 1.00 27.43 B
CA 02373918 2002-02-28
71
ATOM 624 0 LYSB 12 -6.268 42.09823.931 1.00 25.99 B
ATOM 625 N ASNB 13 -5.913 42.80126.048 1.00 25.85 B
ATOM 626 CA ASNB 13 -4.502 42.44626.073 1.00 25.52 B
ATOM 627 CB ASNB 13 -3.628 43.69826.042 1.00 26.55 B
ATOM 628 CG ASNB 13 -3.987 44.62724.882 1.00 29.83 B
ATOM 629 OD1 ASNB 13 -4.218 44.17023.762 1.00 28.51 B
ATOM 630 ND2 ASNB 13 -4.019 45.92925.142 1.00 28.66 B
ATOM 631 C ASNB 13 -4.256 41.68627.369 1.00 25.56 B
ATOM 632 0 ASNB 13 -4.968 41.88528.377 1.00 22.74 B
10ATOM 633 N VALB 14 -3.272 40.79427.337 1.00 23.42 B
ATOM 634 CA VALB 14 -2.913 40.00928.515 1.00 24.09 B
ATOM 635 CB VALB 14 -3.700 38.71228.574 1.00 25.31 B
ATOM 636 CG1 VALB 14 -3.464 37.90227.320 1.00 28.72 B
ATOM 637 CG2 VALB 14 -3.270 37.90729.789 1.00 28.09 B
15ATOM 638 C VALB 14 -1.426 39.70028.432 1.00 25.36 B
ATOM 639 0 VALB 14 -0.856 39.59027.329 1.-0023.77 B
ATOM 640 N GLYB 15 -0.781 39.60529.583 1.00 2'4.37 B
ATOM 641 CA GLYB 15 0.633 39.29629.579 1.00 26.32 B
ATOM 642 C GLYB 15 1.067 38.76630.932 1.00 26.65 B
20ATOM 643 0 GLYB 15 0.325 38.85631.921 1.00 23.77 B
ATOM 644 N TRPB 16 2.251 38.16930.975 1.00 24.82 B
ATOM 645 CA TRPB 16 2.774 37.68532.243 1.00 24.33 B
ATOM 646 CB TRPB 16 2:146 36.34032.618 1.00 24.57 B
ATOM 647 CG TRPB 16 2.571 35.18731.758 1.00 28.68 B
25ATOM 648 CD2 TRPB 16 1.707 34.28631.068 1.00 29.83 B
ATOM 649 CE2 TRPB 16 2.523 33.28830.474 1.00 32.16 B
ATOM 650 CE3 TRPB 16 0.319 34.22330.885 1.00 31.66 B
ATOM 651 CD1 TRPB 16 3.849 34.72031.563 1.00 28.36 B
ATOM 652 NE1 TRPB 16 3.826 33.57230.797 1.00 30.79 B
30ATOM 653 CZ2 TRPB 16 1.989 32.23429.719 1.00 32.79 B
ATOM 654 CZ3 TRPB 16 -0.209 33.17630.136 1.00 32.52 B
ATOM 655 CH2 TRPB 16 0.624 32.20029.561 1.00 33.65 B
ATOM 656 C TRPB 16 4:286 37.55332.173 1.00 22.17 B
ATOM 657 0 TRPB 16 4.885 37.61631.103 1.00 19.67 B
35ATOM 658 N ALAB 17 4.896 37.40633.335 1.00 20.00 B
ATOM 659 CA ALAB 17 6.330 37.22633.431 1.00 20.97 B
ATOM 660 CB ALAB 17 7.008 38.54933.720 1.00 19.52 B
ATOM 661 C ALAB 17 6.429 36.29634.629 1.00 22.18 B
ATOM 662 0 ALAB 17 5.936 36.62935.707 1.00 20.03 B
40ATOM 663 N THRB 18 6.994 35.10634.442 1:00 21.90 B
ATOM 664 CA THRB 18 7.117 34.18335.556 1.00 23.51 B
ATOM 665 CB THRB 18 6.289 32.89935.317 1.00 24.62 B
ATOM 666 OG1 THRB 18 6.757 32.24334.135 1.00 24.95 B
ATOM 667 CG2 THRB 18 4.788 33.23135.129 1.00 24 B
61
45ATOM 668 C THRB 18 8.600 33.81735.753 1.00 . B
25.91
ATOM 669 0 THRB 18 9.398 33.85234.797 1.00 22.59 B
ATOM 670 N GLNB 19 8.973 33.52036.992 1.00 26.80 B
ATOM 671 CA GLNB 19 10.346 33.13037.281 1.00 33.83 B
ATOM 672 CB GLNB 19 10.931 33.94638.435 1.00 36 B
03
50ATOM 673 CG GLNB 19 10.967 35.45138.198 1.00 . B
40.64
ATOM 674 CD GLNB 19 9.580 36.08438.255 1.00 44.69 B
ATOM 675 OE1 GLNB 19 8.731 35.66839.061 1.00 47.37 B
ATOM 676 NE2 GLNB 19 9.342 37.10037.412 1.00 45.52 B
ATOM 677 C GLNB 19 10.348 31.65437.648 1.00 36 B
07
55ATOM 678 0 GLNB 19 9.979 30.81336.824 1.00 . B
40.72
ATOM 679 N LEUB 20 10.741 31.31138.867 1.00 35.52 B
ATOM 680 CA LEUB 20 10.780 29.90039.225 1.00 33.73 B
ATOM 681 CB LEUB 20 11.898 29.62840.241 1.00 36.64 B
ATOM 682 CG LEUB 20 12.300 28.14740.301 1.00 38 B
88
60ATOM 683 CD1 LEUB 20 13.121 27.83539.050 1.00 . B
40.32
ATOM 684 CD2 LEUB 20 13.121 27.84341.542 1.00 41.16 B
ATOM 685 C LEU 20 9.438 29.44639.793 1.00 32:93 B
B
CA 02373918 2002-02-28
72
ATOM 686 0 LEUB 20 8.748 28.62439.192 1.00 34.16 B
ATOM 687 N THRB 21 9.058 29.98940.942 1.00 29.35 B
ATOM 688 CA THRB 21 7.788 29.61441.572 1.00 30.06 B
ATOM 689 CB THRB 21 8.028 29.09242.978 1.00 2:8.17 B
ATOM 690 OG1 THRB 21 8:805 30.06343.689 1.00 30.80 B
ATOM 691 CG2 THRB 21 8.807 27.75442.950 1.00 29.30 B
ATOM 692 C THRB 21 6.842 30.81741.712 1.00 28.78 B
ATOM 693 0 THRB 21 5.830 30.74942.420 1.00 29.23 B
ATOM 694 N SERB 22 7.180 31.92441.073 1.00 28.03 B
ATOM 695 CA SERB 22 6.337 33.09841.213 1.00 27.21 B
ATOM 696 CB SERB 22 6.954 3 4.04242.244 1.00 2 8.04 B
ATOM 697 OG SERB 22 8.181 34.54241.757 1.00 32.79 B
ATOM 698 C SERB 22 6.150 33.79839.904 1.00 25.42 B
ATOM 699 0 SERB 22 6.892 33.55238.952 1.00 23.76 B
ATOM 700 N GLYB 23 5.155 34:67839.848 1.00 23.40 B
ATOM 701 CA GLYB 23 4.918 35.39738.616 1.00 23.53 B
ATOM 702 C GLYB 23 3.945 36.54038.790 1.00 22.85 B
ATOM 703 O GLYB 23 3.347 36.70539.858 1.00 19.43 B
ATOM 704 N ALAB 24 3.803 37.33637.734 1.00 22.99 B
ATOM 705 CA ALAB 24 2.872 38.44237.729 1.00 24.00 B
ATOM 706 CB ALAB 24 3.630 39.76837.827 1.00 23.97 B
ATOM 707 C ALAB 24 2.138 38.32336.403 1.00 24.31 B
ATOM 708 0 ALAB 24 2.732 37,94235.377 1.00 23.13 B
ATOM 709 N VALB 25 0.837 38.59436.448 1.00 2'4.53 B
ATOM 710 CA VALB 25 -0.047 38.55035.281 1.00 25.48 B
ATOM 711 CB VALB 25 -1.244 37.60535.490 1.00 28.33 B
ATOM 712 CG1 VALB 25 -2:184 37.66834.255 1.00 28.58 B
ATOM 713 CG2 VALB 25 -0.770 36:19635.726 1.00 30.81 B
ATOM 714 C VALB 25 -0.650 39.93635.120 1.00 26.05 B
ATOM 715 0 VALB 25 -1.015 40:56036.119 1.00 24.83 B
ATOM 716 N TRPB 26 -0.737 40.41633.884 1.00 24.31 B
ATOM 717 CA TRPB 26 -1.322 41.71933.591 1.00 26.24 B
ATOM 718 CB TRPB 26 -0.268 42.64032.978 1.00 30.96 B
ATOM 719 CG TRPB 26 -0.844 4 3.77032.197 1.00 37.86 B
ATOM 720 CD2 TRPB 26 -0.868 43.90130.761 1.00 40.27 B
ATOM 721 CE2 TRPB 26 -1.479 45.14930.460 1.00 41.09 B
ATOM 722 CE3 TRPB 26 -0.429 43.08529.702 1.00 41.59 B
ATOM 723 CD1 TRPB 26 -1.435 44.90932.695 1.00 39.70 B
ATOM 724 NE1 TRPB 26 -1.817 45.74431.650 1.00 41.13 B
ATOM 725 CZ2 TRPB 26 -1.656 45.59929.144 1.00 42.05 B
ATOM 726 C23 TRPB 26 -0.606 43.53328.393 1.00 42.03 B
ATOM 727 CH2 TRPB 26 -1.215 44.78428.128 1:00 42.46 B
ATOM 728 C TRPB 26 -2.476 41:51132.584 1.00 25.57 B
ATOM 729 0 TRPB 26 -2.385 40.66931.664 1.00 21.95 B
ATOM 730 N VALB 27 -3.555 42.27032.757 1.00 23.60 B
ATOM 731 CA VALB 27 -4.712 42.17031.875 1.00 22.60 B
ATOM 732 CB VALB 27 -5.863 41.38632.532 1.00 23.81 B
ATOM 733 CG1 VALB 27 -7.011 41.26031.539 1.00 22.72 B
ATOM 734 CG2 VALB 27 -5.377 39.99833.014 1.00 25.00 B
ATOM 735 C VALB 27 -5.278 43.55831.583 1.00 24.68 B
ATOM 736 0 VALB 27 -5.430 44.37132.495 1.00 23.54 B
ATOM 737 N GLNB 28 -5.590 43.82930:322 1.00 24.45 B
ATOM 738 CA GLNB 28 -6.203 45.10029.949 1.00 26.46 B
ATOM 739 CB GLNB 28 -5.369 45.83028.913 1.00 30.46 B
ATOM 740 CG GLNB 28 -4.608 46.99229.461 1.00 38.64 B
ATOM 741 CD GLNB 28 -3.935 47,79528.361 1.00 43.10 B
ATOM 742 OE1 GLNB 28 -4.569 48.15627.365 1.00 46.00 B
ATOM 743 NE2 GLNB 28 -2.649 48.08828.540 1.00 44.70 B
ATOM 744 C GLNB 28 -7.548 44.74529.326 1.00 24.08 B
ATOM 745 O GLNB 28 -7.620 43.90028.430 1.00 24.15 B
ATOM 746 N PHEB 29 -8.610 45.38029.795 1.00 22.39 B
ATOM 747 CA PHEB 29 -9:936 45.11529.251 1.00 21.79 B
CA 02373918 2002-02-28
73
ATOM 748 CB PHEB 29 -10.966 45.09830.382 1.00 20.55 B
ATOM 749 CG PHEB 29 -10.716 44.01931.385 1.00 21.12 B
ATOM 750 CD1 PHEB 29 -10.012 44.28332.540 1.00 21.25 B
ATOM 751 CD2 PHEB 29 -11.143 42.71131.141 1.00 22.04 B
ATOM 752 CE1 PHEB 29 -9.724 43.26133.458 1.00 21.96 B
ATOM 753 CE2 PHEB 29 -10.865 41.68632.045 1.00 21.64 B
ATOM 754 CZ PHEB 29 -10.155 41.95933.208 1.00 22.73 B
ATOM 755 C PHEB 29 -10.277 46.17028.204 1.00 22.83 B
ATOM 756 0 PHEB 29 -9.622 47.21628.129 1.00 23.60 B
ATOM 757 N ASNB 30 -11.303 45.90627.403 1.00 24.69 B
ATOM 758 CA ASNB 30 -11.691 46.82826.352 1.00 27.19 B
ATOM 759 CB ASNB 30 -12.741 46.17825.463 1.00 29.82 B
ATOM 760 CG ASNB 30 -14.071 45.99526.163 1.00 31.05 B
ATOM 761 OD1 ASNB 30 -14.140 45.55027.306 1.00 32.99 B
ATOM 762 ND2 ASNB 30 -15.148 46.34425.467 1.00 35.35 B
ATOM 763 C ASNB 30 -12.205 48.16226.892 1.00 27.09 B
ATOM 764 0 ASNB 30 -12.228 49.14326.167 1.00 27.50 B
ATOM 765 N ASPB 31 -12.593 48.21228.164 1.00 27.50 B
ATOM 766 CA ASPB 31 -13.098 49.47128.728 1.00 28.13 B
ATOM 767 CB ASPB 31 -14.125 49.21629.852 1.00 24.72 B
ATOM 768 CG ASPB 31 -13.528 48.51631.050 1.00 24.30 B
ATOM 769 OD1 ASPB 31 -12.329 48.15031.021 1.00 22.63 B
ATOM 770 OD2 ASPB 31 -14.263 48.33132.037 1.00 24.53 B
ATOM 771 C ASPB 31 -11.948 50.31529.246 1.00 27.69 B
ATOM 772 0 ASPB 31 -12.161 51.36929.837 1.00 28.05 B
ATOM 773 N GLYB 32 -10.723 49.84529.018 1.00 27.51 B
ATOM 774 CA GLYB 32 -9.553 50.58829,461 1.00 2.6.87 B
ATOM 775 C GLYB 32 -9.062 50.28130.875 1.00 25.54 B
ATOM 776 0 GLYB 32 -8.039 50.82231.299 1.00 27.40 B
ATOM 777 N SERB 33 -9.772 49.44531.619 1.00 23.05 B
ATOM 778 CA SERB 33 -9.317 49.13032.972 1.00 22.45 B
ATOM 779 CB SERB 33 -10.454 48.58933:806 1.00 19.16 B
ATOM 780 OG SERB 33 -10.999 47.39533.238 1.00 20.75 B
ATOM 781 C SERB 33 -8.187 48.10432.884 1.00 21:94 B
ATOM 782 0 SERB 33 -8.013 47.45431.828 1.00 20.92 B
ATOM 783 N GLNB 34 -7.422 47.97533.967 1.00 22.53 B
ATOM 784 CA GLNB 34 -6.262 47.06434.018 1.00 24.67 B
ATOM 785 CB GLNB 34 -4.956 47.82833.838 1.00 27.02 B
ATOM 786 CG GLNB 34 -4.877 48.73632.647 1.00 34.24 B
ATOM 787 CD GLNB 34 -3.546 49.45032.589 1.00 37.10 B
ATOM 788 OEl GLNB 34 -2.487 48.82532.361 1.00 37.26 B
ATOM 789 NE2 GLNB 34 -3.575 50.76832.816 1.00 38.53 B
ATOM 790 C GLNB 34 -6.127 46.35035.358 1.00 25.48 B
ATOM 791 0 GLNB 34 -6.407 46.93836.421 1.00 24.70 B
ATOM 792 N LEUB 35 -5.685 45.09535.304 1.00 22.93 B
ATOM 793 CA LEUB 35 -5.435 44.28436.505 1.00 23.57 B
ATOM 794 CB LEUB 35 -6.301 43.01636.507 1.00 24.87 B
ATOM 795 CG LEUB 35 -7.740 43.07436.983 1.00 26.23 B
ATOM 796 CD1 LEUB 35 -8.511 41.84136.512 1.00 26.00 B
ATOM 797 CD2 LEUB 35 -7.750 43.18738.513 1.00 23.79 B
ATOM 798 C LEUB 35 -3.975 43.82636.478 1.00 24.00 B
ATOM 799 O LEUB 35 -3.497 43.34235.445 1.00 22.60 B
ATOM 800 N VALB 36 -3.252 44.02237.578 1.00 23.46 B
ATOM 801 CA VALB 36 -1.887 43.51537.684 1.00 24.10 B
ATOM 802 CB VALB 36 -0.840 44.61937.903 1.00 24.21 B
ATOM 803 CG1 VALB 36 0.549 43.98338.042 1.00 26.16 B
ATOM 804 CG2 VALB 36 -0.836 45.57836.742 1.00 24.66 B
ATOM 805 C VALB 36 -1.969 42.61438.923 1.00 25.01 B
ATOM 806 0 VALB 36 -2.338 43.08340.022 1.00 22.79 B
ATOM 807 N METB 37 -1.689 41.31738.738 1.00 23.05 B
ATOM 808 CA METB 37 -1.783 40.34039.829 1.00 22.68 B
ATOM 809 CB METB 37 -2.847 39.29539.491 1.00 22.71 B
CA 02373918 2002-02-28
74
ATOM 810 CG METB 37 -3.996 39.93338.756 1.00 26.04 B
ATOM 811 SD METB 37 -5.501 39.06838.822 1.00 26.70 B
ATOM 812 CE METB 37 -5.327 37.92737.565 1.00 26.34 B
ATOM 813 C METB 37 -0.478 39.62940.099 1.00 2'2.50 B
ATOM 814 0 METB 37 0.183 39.16039.164 1.00 23.17 B
ATOM 815 N GLNB 38 -0.118 39.54141.373 1.00 21.09 B
ATOM 816 CA GLNB 38 1.110 38.86441.790 1.00 21.56 B
ATOM 817 CB GLNB 38 1.715 39.58443.003 1.00 23.88 B
ATOM 818 CG GLNB 38 2.308 40.99042.679 1.00 2'6.47 B
ATOM 819 CD GLNB 38 3.393 40.93541.597 1.00 29.08 B
ATOM 820 OE1 GLNB 38 4.103 39.93541.474 1.00 29.45 B
ATOM 821 NE2 GLNB 38 3.529 42.00940.820 1.00 28.64 B
ATOM 822 C GLNB 38 0.629 37.46242.177 1.00 21.49 B
ATOM 823 O GLNB 38 -0.401 37.32942.818 2.00 19.42 B
ATOM 824 N ALAB 39 1.371 36.42641.805 1.00 19.11 B
ATOM 825 CA ALAB 39 0.946 35.06742.089 1.00 20.63 B
ATOM 826 CB ALAB 39 0.194 34.48340.874 1.00 2'0.95 B
ATOM 827 C ALAB 39 2.129 34.18542.439 1.00 21.22 B
ATOM 828 0 ALAB 39 3.292 34.54842.211 1.00 20.65 B
ATOM 829 N GLYB 40 1.833 33.03343.023 1.00 21.29 B
ATOM 830 CA GLYB 40 2.910 32.12543.382 1.00 22.60 B
ATOM 831 C GLYB 40 2.422 30.70643.549 1.00 22.77 B
ATOM 832 O GLYB 40 1.265 30.44843.914 1.00 21.33 B
ATOM 833 N VALB 41 3.322 29.77243.270 1.00 23.45 B
ATOM 834 CA VALB 41 3.037 28.35943.424 1.00 22.65 B
ATOM 835 CB VALB 41 4.088 27.54042.681 1.00 23.98 B
ATOM 836 CG1 VALB 41 3.925 26.05642.983 1.00 23.94 B
ATOM 837 CG2 VALB 41 3.935 27.80341.183 1.00 25.98 B
ATOM 838 C VALB 41 3.108 28.07444.910 1.00 21.21 B
ATOM 839 0 VALB 41 4.054 28.46645.557 1.00 20.44 B
ATOM 840 N SERB 42 2.111 27.38445.453 1.00 22.01 B
ATOM 841 CA SERB 42 2.081 27.09546.882 1.00 21.18 B
ATOM 842 CB SERB 42 0.741 27.55947.448 1.00 19.17 B
ATOM 843 OG SERB 42 -0.294 26.96846.698 1.00 21.34 B
ATOM 844 C SERB 42 2.317 25.60647.203 1.00 24.43 B
ATOM 845 O SERB 42 2.600 25.25848.354 1.00 25.28 B
ATOM 846 N SERB 43 2.126 24.73246.213 1.00 24.56 B
ATOM 847 CA SERB 43 2.411 23.29646.374 1.00 24.94 B
ATOM 848 CB SERB 43 1.241 22.47846.977 1.00 23.50 B
ATOM 849 OG SERB 43 0.016 22.63146.302 1.00 28.57 B
ATOM 850 C SERB 43 2.849 22.69945.043 1.00 26.29 B
ATOM 851 O SERB 43 2.355 23.06943.969 1.00 24.41 B
ATOM 852 N ILEB 44 3.811 21.78045.120 1.00 25.28 B
ATOM 853 CA ILEB 44 4.337 21,11543.947 1.00 24.80 B
ATOM 854 CB ILEB 44 5.788 21.58043.673 1.00 27.42 B
ATOM 855 CG2 ILEB 44 6.427 20.73142.572 1.00 26.96 B
ATOM 856 CG1 ILEB 44 5.780 23.06243.293 1.00 26.37 B
ATOM 857 CD1 ILEB 44 7.143 23.65443.008 1.00 28.02 B
ATOM 858 C ILEB 44 4.285 19.61744.201 1.00 26 B
01
ATOM 859 O ILEB 44 4.629 19.14545.294 1.00 . B
23.90
ATOM 860 N SERB 45 3.816 18.89043.195 1.00 25.60 B
ATOM 861 CA SERB 45 3.685 17.44843.257 1.00 25.92 B
ATOM 862 CB SERB 45 2.206 17.09543.212 1.00 28.69 B
ATOM 863 OG SERB 45 1.981 15.69743.280 1.00 33 B
00
ATOM 864 C SERB 45 4.440 16.89142.044 1.00 . B
26.07
ATOM 865 0 SERB 45 3.989 17.04340.895 1.00 26.72 B
ATOM 866 N TYRB 46 5.615 16.30542.302 1.00 23.13 B
ATOM 867 CA TYRB 46 6.459 15.72141.263 1.00 22.07 B
ATOM 868 CB TYRB 46 7.947 15.94041.573 1.00 21 B
10
ATOM 869 CG TYRB 46 8.887 15.28940.560 1.00 . B
21.47
ATOM 870 CD1 TYRB 46 9.105 15.87439.324 1.00 20.80 B
ATOM 871 CE1 TYRB 46 9.986 15.32038.396 1.00 22.20 B
CA 02373918 2002-02-28
ATOM 872 CD2 TYRB 46 9.580 14.09740.860 1.00 22.24 B
ATOM 873 CE2 TYRB 46 10.476 13.52339.938 1.00 21.77 B
ATOM 874 CZ TYRB 46 10:668 14.14738.704 1.00 22.48 B
ATOM 875 OH TYRB 46 11.518 13.61837.763 1.00 22.53 B
5 ATOM 876 C TYRB 46 6.245 14.21341.140 1.00 23.70 B
ATOM 877 O TYRB 46 6.366 13.46842.127 1.00 24.10 B
ATOM 878 N THRB 47 5.949 13.75139.935 1.00 22.63 B
ATOM 879 CA THRB 47 5.784 12.31039.730 1.00 23.41 B
ATOM 880 CB THRB 47 4.451 11.99039.079 1.00 23.58 B
10 ATOM 881 OG1 THRB 47 3.407 12.37939.977 1.00 25.78 B
ATOM 882 CG2 THRB 47 4.332 10.47838.800 1.00 2'4.55
B
ATOM 883 C THRB 47 6.913 11.86638.821 1.00 21.48 B
ATOM 884 0 THRB 47 7.002 12.31737.679 1.00 21.40 B
ATOM 885 N SERB 48 7.786 11.00539.340 1.00 20.62 B
15 ATOM 886 CA SERB 48 8.944 10.52838.588 1.00 19.68 B
ATOM 887 CB SERB 48 9.837 9.668 39.480 1.00 19.79 B
ATOM 888 OG SERB 48 9.147 8.463 39.856 1.00 20.44 B
ATOM 889 C SERB 48 8.517 9.706 37.394 1.00 20.21 B
ATOM 890 O SERB 48 7.360 9.286 37.300 1.00 20.77 B
20 ATOM 891 N PROB 49 9.453 9.429 36.475 1.00 20.80 B
ATOM 892 CD PROB 49 10.839 9.921 36.382 1.00 19.25 B
ATOM 893 CA PROB 49 9.108 8.635 35.293 1.00 21.42 B
ATOM 894 CB PROB 49 10.434 8.535 34.547 1.00 19.67 B
ATOM 895 CG PROB 49 11.072 9.870 34.879 1.00 1 9.88
B
25 ATOM 896 C PROB 49 8.548 7.286 35.677 1.00 22.31 B
ATOM 897 O PROB 49 7.734 6.724 34.938 1.00 22.96 B
ATOM 898 N ASPB 50 8.957 6.771 36.834 1.00 22.40 B
ATOM 899 CA ASPB 50 8.466 5.474 37.308 1.00 24.50 B
ATOM 900 CB ASPB 50 9.561 4.738 38.096 1.00 24.53 B
30 ATOM 901 CG ASPB 50 10.661 4.197 37.181 1.00 23.14 B
ATOM 902 OD1 ASPB 50 10.388 3.260 36.395 1.00 25.84 B
ATOM 903 OD2 ASPB 50 11.788 4.726 37.217 1.00 2T.10 B
ATOM 904 C ASPB 50 7.167 5.537 38.134 1.00 26.54 B
ATOM 905 0 ASPB 50 6.720 4.526 38.719 1.00 24.08 B
35 ATOM 906 N GLYB 51 6.558 6.722 38.180 1.00 25.82 B
ATOM 907 CA GLYB 51 5.291 6.856 38.873 1.00 26.67 B
ATOM 908 C GLYB 51 5.327 7.099 40.366 1.00 28.06 B
ATOM 909 0 GLYB 51 4.319 6.892 41.031 1:00 30.08 B
ATOM 910 N GLNB 52 6.459 7.497 40.922 1.00 27.83 B
40 ATOM 911 CA GLNB 52 6.486 7.770 42.361 1.00 29.96 B
ATOM 912 CB GLNB 52 7.822 7.330 42.966 1.00 32.90 B
ATOM 913 CG GLNB 52 8.182 5.864 42.564 1.00 38.75 B
ATOM 914 CD GLNB 52 6.963 4.910 42.669 1.00 41.93 B
ATOM 915 OE1 GLNB 52 6.482 4.599 43.779 1.00 43.80 B
45 ATOM 916 NE2 GLNB 52 6.446 4.466 41.507 1.00 42.57 B
ATOM 917 C GLNB 52 6.262 9.279 42.581 1.00 28.82 B
ATOM 918 0 GLNB 52 6.939 10.11941.966 1.00 27.16 B
ATOM 919 N THRB 53 5.325 9.612 43.465 1.00 28.03 B
ATOM 920 CA THRB 53 4.991 11.02143.733 1.00 27.41 B
50 ATOM 921 CB THRB 53 3.455 11.22443.667 1.00 26.41 B
ATOM 922 OG1 THRB 53 3.014 10.87842.347 1.00 24.55 B
ATOM 923 CG2 THRB 53 3.066 12.72143.946 1.00 27.48 B
ATOM 924 C THRB 53 5.527 11.56145.060 1.00 26.97 B
ATOM 925 0 THRB 53 5.439 10.91146.098 1.00 26.40 B
55 ATOM 926 N THRB 54 6.109 12.75144.993 1.00 25.91 B
ATOM 927 CA THRB 54 6.654 13.43546.148 1.00 25.43 B
ATOM 928 CB THRB 54 8.185 13.54346.048 1.00 26.30 B
ATOM 929 OG1 THRB 54 8.731 12.21746.017 1.00 27.51 B
ATOM 930 CG2 THRB 54 8.761 14.27047.249 1.00 26.98 B
60 ATOM 931 C THRB 54 6.032 14.81146.119 1.00 24.29 B
ATOM 932 O THRB 54 6.058 15.49445.085 1,00 21.45 B
ATOM 933 N ARGB 55 5.450 15.20247.244 1.00 24.59 B
CA 02373918 2002-02-28
76
ATOM 934 CA ARGB 55 4.791 16.49847.343 1.00 28.29 B
ATOM 935 CB ARGB 55 3.382 16.30747.906 1.00 3;0.45 B
ATOM 936 CG ARGB 55 2.547 15.39547:020 1.00 36.62 B
ATOM 937 CD ARGB 55 1.134 15.16547.550 1.00 40.16 B
ATOM 938 NE ARGB 55 0.425 14.19146.728 1.00 42.96 B
ATOM 939 CZ ARGB 55 0.574 12.87146:832 1.00 45.47 B
ATOM 940 NH1 ARGB 55 1.409 12.35447.736 1.00 45.46 B
ATOM 941 NH2 ARGB 55 -0.107 12.06346.019 1.00 45.66 B
ATOM 942 C ARGB 55 5.565 17.48848.195 1.00 27.52 B
10ATOM 943 0 ARGB 55 6.225 17.11249.167 1.00 27.20 B
ATOM 944 N TYRB 56 5.513 18.75447.803 1.00 27.30 B
ATOM 945 CA TYRB 56 6.194 19.79448.548 1.00 28.29 B
ATOM 946 CB TYRB 56 7.414 20.31247.804 1.00 29.22 B
ATOM 947 CG TYRB 56 8.406 19.23447.446 1.00 33.48 B
15ATOM 948 CD1 TYRB 56 8.220 18.44346.307 1.00 32.53 B
ATOM 949 CE1 TYRB 56 9.167 17.49045.932 1.00 35.51 B
ATOM 950 CD2 TYRB 56 9.560 19.03548.216 1.00 33.59 B
ATOM 951 CE2 TYRB 56 10.514 18.07347.853 1.00 37.05 B
ATOM 952 CZ TYRB 56 10.312 17.31346.705 1.00 36.68 B
20ATOM 953 OH TYRB 56 11.279 16.42646.287 1.00 39.21 B
ATOM 954 C TYRB 56 5.242 20.94348.771 1.00 28:88 B
ATOM 955 0 TYRB 56 4.520 21.36347.858 1.00 28:25 B
ATOM 956 N GLYB 57 5.228 21.40850.014 1.00 28.70 B
ATOM 957 CA GLYB 57 4.407 22.52450.412 1.00 26.92 B
25ATOM 958 C GLYB 57 5.242 23.77750.511 1.00 27.44 B
ATOM 959 0 GLYB 57 6.460 23.75950.327 1.00 25.48 B
ATOM 960 N GLUB 58 4.562 24.87250.838 1.00 28.85 B
ATOM 961 CA GLUB 58 5.170 26.19550.944 1.00 31.01 B
ATOM 962 CB GLUB 58 4.075 27:20951.289 1.00 32.60 B
30ATOM 963 CG GLUB 58 4.296 28.53950.685 1.00 36.52 B
ATOM 964 CD GLUB 58 3.023 29.35350.618 1.00 37.61 B
ATOM 965 OE1 GLUB 58 3.041 30.32549.847 1.00 39.19 B
ATOM 966 OE2 GLUB 58 2.030 29.01651.315 1.00 35.73 B
ATOM 967 C GLUB 58 6.289 26.29751.961 1.00 29.67 B
35ATOM 968 O GLUB 58 7.207 27.08451.795 1.00 28.65 B
ATOM 969 N ASNB 59 6.207 25.50053.017 1.00 29.60 B
ATOM 970 CA ASNB 59 7 . 217 25 54.072 1. 30 . B
. 00 67
507
ATOM 971 CB ASNB 59 6.558 25.09555.400 1.00 31.78 B
ATOM 972 CG ASNB 59 7.436 25.37456.616 1.00 34.02 B
40ATOM 973 OD1 ASNB 59 7.427 24.60357.590 1.00 36.16 B
ATOM 974 ND2 ASNB 59 8.163 26.49056.588 1.00 32.45 B
ATOM 975 C ASNB 59 8.388 24.54353:772 1.00 30.66 B
ATOM 976 0 ASNB 59 9.262 24.37054.609 1.00 28.83 B
ATOM 977 N GLUB 60 8.405 23.91252.596 1.00 31.84 B
45ATOM 978 CA GLUB 60 9.484 22.95952.263 1.00 33.09 B
ATOM 979 CB GLUB 60 8.881 21.60651.857 1.00 33.04 B
ATOM 980 CG GLUB 60 8.009 20.95852.956 1.00 33.74 B
ATOM 981 CD GLUB 60 7.326 19.64752.512 1.00 37.38 B
ATOM 982 OE1 GLUB 60 6.136 19.67152.091 1.00 36.26 B
50ATOM 983 OE2 GLUB 60 7.989 18.58752.584 1.00 37.34 B
ATOM 984 C GLUB 60 10.407 23.45051.155 1.00 34.34 B
ATOM 985 0 GLUB 60 10.012 24.24950.301 1.00 34.69 B
ATOM 986 N LYSB 61 11.657 22.99951.185 1.00 34.58 B
ATOM 987 CA LYSB 61 12.618 23.37850.155 1.00 34.67 B
55ATOM 988 CB LYSB 61 14.023 23.47450.757 1.00 37.32 B
ATOM 989 CG LYSB 61 15.136 23.58649.719 1.00 40.98 B
ATOM 990 CD LYSB 61 16.441 24.12250.309 1.00 44.39 B
ATOM 991 CE LYSB 61 16.911 23.34451.533 1.00 46.53 B
ATOM 992 NZ LYSB 61 18.090 24.02852.184 1.00 48.46 B
60ATOM 993 C LYSB 61 12,591 22.33349.025 1.00 33.73 B
ATOM 994 0 LYSB 61 12.375 21.13949.271 1.00 32.48 B
ATOM 995 N LEUB 62 12.784 22.79347.791 1.00 31.87 B
CA 02373918 2002-02-28
77
ATOM 996 CA LEUB 62 12.779 21.91746.622 1.00 32.06 B
ATOM 997 CB LEUB 62 12.216 22.64545.390 1.00 30.85 B
ATOM 998 CG LEUB 62 10.758 23.10445.390 1.00 32.13 B
ATOM 999 CD1 LEUB 62 10.506 23.97444.178 1.00 31.87 B
ATOM 1000 CD2 LEUB 62 9:845 21.87845.365 1.00 31.68 B
ATOM 1001 C LEUB 62 14.193 21.47146.274 1.00 32.22 B
ATOM 1002 0 LEUB 62 15:139 22.24646.421 1.00 31.77 B
ATOM 1003 N PROB 63 14.349 20.21245.813 1.00 32.35 B
ATOM 1004 CD PROB 63 13.289 19.18445.816 1.00 33.22 B
10ATOM 1005 CA PROB 63 15.639 19.63745.416 1.00 32.16 B
ATOM 1006 CB PROB 63 15.319 18.16345.156 1.00 32.18 B
ATOM 1007 CG PROB 63 14.067 17.91845.977 1.00 33.74 B
ATOM 1008 C PROB 63 16.027 20.33044.131 1.00 32.29 B
ATOM 1009 0 PROB 63 15.142 20.81943.403 1.00 30.11 B
15ATOM 1010 N GLUB 64 17.333 20.35343.832 1.00 32.20 B
ATOM 1011 CA GLUB 64 17.840 21.00242.619 1.00 31.44 B
ATOM 1012 CB GLUB 64 19:372 20.97742.567 1.00 33.66 B
ATOM 1013 CG GLUB 64 20.040 21.98443.496 1.00 39.13 B
ATOM 1014 CD GLUB 64 19.571 23.41443.252 1.00 40.51 B
20ATOM 1015 OE1 GLUB 64 19.507 23.82842.065 1.00 41.69 B
ATOM 1016 OE2 GLUB 64 19.273 24.12044.250 1.00 44.35 B
ATOM 1017 C GLUB 64 17.331 20.43341.313 1.00 30.81 B
ATOM 1018 0 GLUB 64 17.141 21.17840.336 1.00 30.24 B
ATOM 1019 N TYRB 65 17.125 19.12341.244 1.00 2 8.06 B
25ATOM 1020 CA TYRB 65 16.654 18.58839.971 1.00 28.85 B
ATOM 1021 CB TYRB 65 16.779 17.05939.937 1.00 28.86 B
ATOM 1022 CG TYRB 65 15.746 16.32940.744 1.00 28.28 B
ATOM 1023 CD1 TYRB 65 14.620 15.78840.124 1.00 30.60 B
ATOM 1024 CE1 TYRB 65 13.701 15.04240.828 1.00 29.77 B
30ATOM 1025 CD2 TYRB 65 15.916 16.11842.106 1.00 27.97 B
ATOM 1026 CE2 TYRB 65 14.989 15.37542.837 1.00 29.71 B
ATOM 1027 CZ TYRB 65 13.890 14.83842.190 1.00 30.50 B
ATOM 1028 OH TYRB 65 12.966 14.09542.883 1.00 30.91 B
ATOM 1029 C TYRB 65 15.209 19.03439.714 1.00 27.21 B
35ATOM 1030 0 TYRB 65 14.775 19.10938.572 1.00 27.93 B
ATOM 1031 N ILEB 66 14.466 19.32740.775 1.00 27.73 B
ATOM 1032 CA LLEB 66 13.088 19.81340.616 1.00 29.70 B
ATOM 1033 CB ILEB 66 12.312 19.78041:951 1.00 30.45 B
ATOM 1034 CG2 ILEB 66 10.896 20.37741.761 1.00 31.18 B
40ATOM 1035 CG1 ILEB 66 12.262 18.34742.500 1.00 33.52 B
ATOM 1036 CD1 ILEB 66 11.287 17.46041.858 1.00 30.53 B
ATOM 1037 C ILEB 66 13.147 21.28040.138 1.00 30.08 B
ATOM 1038 0 ILEB 66 12.441 21.66539.215 1.00 30.26 B
ATOM 1039 N LYSB 67 14.001 22.08440.766 1.00 30.53 B
45ATOM 1040 CA LYSB 67 14.145 23.50040.411 1.00 33.59 B
ATOM 1041 CB LYSB 67 15.194 24.18541.290 1.00 33.18 B
ATOM 1042 CG LYSB 67 15.064 23.88142.786 1.00 37.68 B
ATOM 1043 CD LYSB 67 14.811 25.14343.630 1.00 40.72 B
ATOM 1044 CE LYSB 67 15.987 26.09543.620 1.00 41.58 B
50ATOM 1045 NZ LYSB 67 17.116 25.64844.477 1.00 45.16 B
ATOM 1046 C LYSB 67 14:570 23.62038.955 1.00 34.20 B
ATOM 1047 O LYSB 67 14.023 24.43538.201 1.00 34.67 B
ATOM 1048 N GLNB 68 15.554 22.81238.569 1.00 33.52 B
ATOM 1049 CA GLNB 68 16.047 22.80137.208 1.00 34.63 B
55ATOM 1050 CB GLNB 68 16.992 21.61837.010 1.00 39.08 B
ATOM 1051 CG GLNB 68 18.215 21.64937.878 1.00 44.05 B
ATOM 1052 CD GLNB 68 19.392 22.24337.150 1.00 47.61 B
ATOM 1053 OE1 GLNB 68 19.400 23.44236.815 1.00 49.22 B
ATOM 1054 NE2 GLNB 68 20.401 21.40536.880 1.00 48.50 B
60ATOM 1055 C GLNB 68 14.859 22.63736.270 1.00 33.72 B
ATOM 1056 0 GLNB 68 14.764 23.29535.234 1.00 34.87 B
ATOM 1057 N LYSB 69 13.947 21.74136.614 1.00 31.19 B
CA 02373918 2002-02-28
78
ATOM 1058 CA LYSB 69 12.803 21.53135.741 1.00 30.30 B
ATOM 1059 CB LYSB 69 12.076 20.23736.126 1.00 27.57 B
ATOM 1060 CG LYSB 69 12.629 19.05735.327 1.00 29.07 B
ATOM 1061 CD LYSB 69 12.272 17.66835.859 1.00 24.54 B
ATOM 1062 CE LYSB 69 12.698 16:60434.821 1.00 25.27 B
ATOM 1063 NZ LYSB 69 12:601 15.21535.374 1.00 20.26 B
ATOM 1064 G LYSB 69 11.882 22:74835.763 1.00 29.46 B
ATOM 1065 0 LYSB 69 11.308 23.11134.734 1.00 30.15 B
ATOM 1066 N LEUB 70 11.765 23.37836.928 1.00 29.80 B
ATOM 1067 CA LEUB 70 10.957 24.58837.081 1.00 33.21 B
ATOM 1068 CB LEUB 70 10.965 25.07638.527 1.00 31.58 B
ATOM 1069 CG LEUB 70 9.957 24.41339.450 1.00 34.15 B
ATOM 1070 CD1 LEUB 70 10.14 24.95440.866 1.00 33.82 B
6
ATOM 1071 CD2 LEUB 70 8.546 24.70638.942 1.00 33.95 B
ATOM 1072 C LEUB 70 11.470 25.71336.192 1.00 32.31 B
ATOM 1073 0 LEUB 70 10.684 26.45935.636 1.00 33.63 B
ATOM 1074 N GLNB 71 12.786 25.83436.053 1.00 35.18 B
ATOM 1075 CA GLNB 71 13.356 26.89935.225 1.00 36.36 B
ATOM 1076 CB GLNB 71 14.887 26.87735.265 1.00 38.30 B
ATOM 1077 CG GLNB 71 15.472 26.77736.654 1.00 43.28 B
ATOM 1078 CD GLNB 71 17.001 26.75936.647 1.00 47.08 B
ATOM 1079 OE1 GLNB 71 17.633 26.35137.626 1.00 49:47 B
ATOM 1080 NE2 GLNB 71 17.599 27.20735.541 1.00 49.59 B
ATOM 1081 C GLNB 71 12.908 26.81733.769 1.00 36.22 B
ATOM 1082 0 GLNB 71 12.818 27.85833.099 1.00 35.03 B
ATOM 1083 N LEUB 72 12.622 25.60633.273 1.00 34.21 B
ATOM 1084 CA LEUB 72 12.201 25.44831.874 1.00 33.60 B
ATOM 1085 CB LEUB 72 12.164 23.96631:455 1.00 34.75 B
ATOM 1086 CG LEUB 72 13.423 23.08931.537 1.00 34.77 B
ATOM 1087 CD1 LEUB 72 13.039 21.60631.382 1.00 34.45 B
ATOM 1088 CD2 LEUB 72 14.401 23.51130.442 1.00 34.92 B
ATOM 1089 C LEUB 72 10.817 26.04331.643 1.00 33.64 B
ATOM 1090 0 LEUB 72 10.400 26.19430.494 1.00 31.65 B
ATOM 1091 N LEUB 73 10.116 26.39232:724 1.00 32.55 B
ATOM 1092 CA LEUB 73 8.764 26.94732.608 1.00 33.85 B
ATOM 1093 CB LEUB 73 7.883 26.43233.754 1.00 35.05 B
ATOM 1094 CG LEUB 73 7.891 24.91233.938 1.00 37.23 B
ATOM 1095 CD1 LEUB 73 7.380 24.55135.344 1.00 38.68 B
ATOM 1096 CD2 LEUB 73 7.056 24.26032.843 1.00 37.27 B
ATOM 1097 C LEUB 73 8.717 28.47632.594 1.00 33.24 B
ATOM 1098 0 LEUB 73 7.746 29.05832.128 1.00 34.63 B
ATOM 1099 N SERB 74 9.764 29.11233.111 1.00 32.48 B
ATOM 1100 CA SERB 74 9.857 30.56333.164 1.00 29.94 B
ATOM 1101 CB SERB 74 11.218 30.96933.719 1:00 30.18 B
ATOM 1102 OG SERB 74 11.398 30.39535.006 1.00 35.73 B
ATOM 1103 C SERB 74 9.650 31.21031.800 1.00 28.75 B
ATOM 1104 0 SERB 74 10.313 30.85330.816 1.00 28.50 B
ATOM 1105 N SERB 75 8.720 32.15831.727 1.00 26.58 B
ATOM 1106 CA SERB 75 8.469 32.85030.463 1.00 23.76 B
ATOM 1107 CB SERB 75 7.521 32.04329.583 1.00 25.25 B
ATOM 1108 OG SERB 75 6.220 31.91530.161 1.00 25.57 B
ATOM 1109 C SERB 75 7.901 34.24030.670 1.00 23.10 B
ATOM 1110 0 SERB 75 7.564 34.63431.779 1:00 19.92 B
ATOM 1111 N ILEB 76 7.875 34.98229.577 1.00 22.80 B
SS ATOM 1112 CA ILEB 76 7.362 36.33029.504 1.00 23.91 B
ATOM 1113 CB ILEB 76 8.500 37.35829.251 1.00 26.24 B
ATOM 1114 CG2 ILEB 76 7.908 38.70928.915 1.00 27.13 B
ATOM 1115 CGl ILEB 76 9.382 37.48330.498 1.00 28.59 B
ATOM 1116 CD1 ILEB 76 10.458 38.55330.372 1.00 33.23 B
ATOM 1117 C ILEB 76 6.479 36.22828.252 1.00 23.80 B
ATOM 1118 0 ILEB 76 6.966 35.83827.166 1.00 22.78 B
ATOM 1119 N LEUB 77 5.194 36.51828.407 1.00 23.20 B
CA 02373918 2002-02-28
79
ATOM 1120 CA LEUB 77 4.239 36.447 27.286 1.00 23.99 B
ATOM 1121 CB LEUB 77 3.244 35.304 27.502 1.00 23.91 B
ATOM 1122 CG LEUB 77 2.153 35.143 26.409 1.00 26.66 B
ATOM 1123 CD1 LEUB 77 1.935 33.666 26.098 1.00 27.56 B
ATOM 1124 CD2 LEUB 77 0.849 35.772 26.875 1.00 26.92 B
ATOM 1125 C LEUB 77 3.468 37.755 27.177 1.00 24.42 B
ATOM 1126 0 LEUB 77 3.112 38.356 28.196 1.00 22.52 B
ATOM 1127 N LEUB 78 3.269 38.218 25.945 1.00 23.94 B
ATOM 1128 CA LEUB 78 2.496 39.425 25.680 1.00 25.25 B
10ATOM 1129 CB LEUB 78 3.403 40.559 25.195 1.00 26.26 B
ATOM 1130 CG LEUB 78 4.448 41.009 26.230 1.00 27.97 B
ATOM 1131 CD1 LEUB 78 5.575 41.856 25.603 1.00 29.40 B
ATOM 1132 CD2 LEUB 78 3.700 41:789 27.297 1.00 31.48 B
ATOM 1133 C LEUB 78 1.541 39.044 24.567 1.00 26.11 B
15ATOM 1134 O LEUB 78 1.973 38.505 23.551 1.00 24.70 B
ATOM 1135 N METB 79 0.249 39.281 24.772 1.00 25.67 B
ATOM 1136 CA METB 79 -0.765 38.995 23.754 1.00 27.63 B
ATOM 1137 CB METB 79 -1.578 37.753 24.106 1.00 28.06 B
ATOM 1138 CG METB 79 -2.443 37.323 22.926 1.00 35.25 B
20ATOM 1139 SD METB 79 -3.262 35.745 23.168 1.00 40.80 B
ATOM 1140 CE METB 79 -2.237 34.993 24.422 1.00 37.94 B
ATOM 1141 C METB 79 -1.715 40.203 23.604 1.00 27.55 B
ATOM 114 0 METB 79 -2.243 40.710 24,602 1.00 24.75 B
2
ATOM 1143 N PHEB 80 -1.906 40.670 22.370 1.00 29.13 B
25ATOM 1144 CA PHEB 80 -2.770 41.822 22.098 1.00 30.08 B
ATOM 1145 CB PHEB 80 -1.965 43.041 21.609 1.00 29.04 B
ATOM 1146 CG PHEB 80 -0.704 43.324 22:396 1.00 30.01 B
ATOM 1147 CD1 PHEB 80 0:462 42.587 22.164 1.00 30.40 B
ATOM 1148 CD2 PHEB 80 -0.672 44.359 23.337 1.00 31.06 B
30ATOM 1149 CE1 PHEB 80 1.647 42.875 22.857 1.00 31.13 B
ATOM 1150 CE2 PHEB 80 0.511 44.665 24.045 1.00 31.57 B
ATOM 1151 CZ PHEB 80 1.675 43:916 23.800 1.00 30.17 B
ATOM 1152 C PHEB 80 -3.770 41.508 20.996 1.00 33.16 B
ATOM 1153 0 PHEB 80 -3.456 40.766 20.050 1.00 30.01 B
35ATOM 1154 N SERB 81 -4.978 42.061 21.112 1.00 35.97 B
ATOM 1155 CA SERB 81 -5.976 41.897 20.048 1.00 40.51 B
ATOM 1156 CB SERB 81 -7.337 42.442 20.494 1.00 39.87 B
ATOM 1157 OG SERB 81 -7.832 41.698 21.591 1.00 40.39 B
ATOM 1158 C SERB 81 -5.438 42.744 18.868 1.00 42.34 B
40ATOM 1159 0 SERB 81 -5.008 43.874 19.063 1.00 44.86 B
ATOM 1160 N ASNB 82 -5.463 42.206 17.654 1.00 44.80 B
ATOM 1161 CA ASNB 82 -4.951 42.914 16.477 1.00 46.28 B
ATOM 1162 CB ASNB 82 -4.259 41.906 15.544 1.00 46.09 B
ATOM 1163 CG ASNB 82 -3.309 42.565 14.537 1.00 46.70 B
45ATOM 1164 OD1 ASNB 82 -2.677 41.874 13.716 1.00 46.56 B
ATOM 1165 ND2 ASNB 82 -3.203 43.891 14.593 1.00 45.41 B
ATOM 1166 C ASNB 82 -6.073 43.653 15.722 1.00 48.50 B
ATOM 1167 0 ASNB 82 -6.410 43.239 14.578 1.00 47.94 B
ATOM 1168 OXT ASNB 82 -6.611 44.640 16.292 1.00 51.85 B
50ATOM 1169 OH2 TIPS 1 1.508 24.728 50.569 1.00 21.12 S
ATOM 1170 OH2 TIPS 2 -4.532 41.128 52.790 1.00 22.93 S
ATOM 1171 OH2 TIPS 3 0.453 33.543 46.169 1.00 21.41 S
ATOM 1172 OH2 TIPS 4 8.870 11.544 43.348 1.00 25.23 S
ATOM 1173 OH2 TIPS 5 -3.457 47.896 37.725 1.00 21.86 S
55ATOM 1174 OH2 TIPS 6 11.989 7.249 38.203 1.00 25.35 S
ATOM 1175 OH2 TIPS 7 1.880 40:091 46.556 1.00 29.15 S
ATOM 1176 OH2 TIPS 8 2.444 35.387 45.395 1.00 29.58 S
ATOM 1177 OH2 TIPS 9 -10.635 6 0.27936.514 1.00 25.71 S
ATOM 1178 OH2 TIPS 10 -5.178 50.690 47.482 1.00 27.75 S
60ATOM 1179 OH2 TIPS 11 5.346 13.571 49.415 1.00 29.60 S
ATOM 1180 ~H2 TIPS 12 11.036 7.211 41.061 1.00 25.43 S
ATOM 1181 OH2 TIPS 13 2.572 14.979 39.851 1.00 25.44 S
CA 02373918 2002-02-28
ATOM 1182 OH2 TIP S 14 -0.581 43.317 42.332 1.0033.47
ATOM 1183 OH2 TIP S 15 -12.815 55.716 40,968 1.0030.28
ATOM 1184 OH2 TIP S 16 -0:965 48.449 38.790 1.0025.80
ATOM 1185 OH2 TIP S 17 -17.201 44.033 35.905 1.0029.81
5 ATOM 1186 OH2 TIP S 18 -2.352 31.966 50.012 1.0021.46
ATOM 1187 OH2 TIP S 19 -12:888 38.321 27.123 1.0031.06
ATOM 1188 OH2 TIP S 20 0.353 20.226 43.760 1.0034.64
ATOM 1189 OH2 TIP S 21 10.886 7.638 30.517 1.0032.87
ATOM 1190 OH2 TIP S 22 -6.652 39.779 46.435 1.0031.11
10 ATOM 1191 OH2 TIP S 23 -9.631 51.555 41.700 1.0029.23
ATOM 1192 OH2 TIP S 24 -6.268 37.267 45.848 1.0030.09
ATOM 1193 OH2 TIP S 25 -10.305 30.700 43.071 1,0034.11
ATOM 1194 OH2 TIP S 26 -13.571 38.030 48.036 1.0038.04
ATOM 1195 OH2 TIP S 27 0.283 14.726 41.020 1.0033.01
15 ATOM 1196 OH2 TIP S 28 16.076 17.853 36.341 1.0032.65
ATOM 1197 OH2 TIP S 29 0.078 30.990 51.479 1.0035.59
ATOM 1198 OH2 TIP S 30 -16.819 48.859 31.799 1.0033.87
ATOM 1199 OH2 TIP S 31 11.178 22.910 27.196 1.0034.94
ATOM 1200 OH2 TIP S 32 4.359 17.883 51.741 1.0034.82
20 ATOM 1201 OH2 TIP S 33 2.022 32.446 50.376 1.0023.48
ATOM 1202 OH2 TIP S 34 19.034 19.266 46.006 1.0035.73
ATOM 1203 OH2 TIP S 35 10.267 32.663 42.312 1.0042.74
ATOM 1204 OH2 TIP S 36 8.286 1.858 35.678 1.0029.10
ATOM 1205 OH2 TIP S 37 -5.005 55.786 42:115 1.0039.52
25 ATOM 1206 OH2 TI S 38 -7.453 59.109 38.085 1.0040.32
P
ATOM 1207 OH2 TIP S 39 -1.225 48.872 43.438 1.0036.53
ATOM 1208 OH2 TIP S 40 5.207 13.791 15.362 1.0041.72
ATOM 1209 0H2 TIP S 41 5.160 10.320 35.567 1.0031.09
ATOM 1210 OH2 TLP S 42 18.752 17.356 43.075 1.0037.74
30 ATOM 1211 OH2 TIP S 43 -18.397 40.367 29.850 1:0048.05
ATOM 1212 OH2 TIP S 44 8.184 37.232 40.970 1.0040.60
ATOM 1213 OH2 TIP S 45 -6.617 31.751 32.951 1.0040.27
ATOM 1214 OH2 TIP S 46 -8.475 47.711 49.511 1.0038.51
ATOM 1215 OH2 TIP S 47 -7.813 36.040 47.740 1,0041.75
35 ATOM 1216 OH2 TIP S 48 6,688 38.985 40.094 1.0037.02
ATOM 1217 OH2 TIP S 49 -12.153 36.097 28.343 1:0043.38
ATOM 1218 OH2 TIP S 50 -19.218 44.577 45.754 1,0039.99
ATOM 1219 OH2 TIP S 51 3.811 7.715 44.758 1.0038.01
ATOM 1220 OH2 TIP S 52 -5.378 33.843 35.965 1.0054.23
40 ATOM 1221 OH2 TIP S 53 -4.266 33.146 37.939 1.0042.29
ATOM 1222 OH2 TIP S 54 -2.398 31.670 38.304 1.0047.47
ATOM 1223 OH2 TIP S 55 2.394 31.399 38.501 1.0049.33
ATOM 1224 OH2 TIP S 56 4.080 30.038 37.667 1.0037.16
ATOM 1225 OH2 TIP S 57 4.352 28.531 35.734 1.0051.95
45 ATOM 1226 OH2 TIP S 58 3.223 29.525 33.569 1.0042.32
END
