Note: Descriptions are shown in the official language in which they were submitted.
CA 02446527 2003-10-23
Method of rational-based drug design using osteocalcin
Feld of the Invention
The invention relates to the crystalline form of osteocalcin ("OC"). The
invention also relates to methods of using the three dimensional structure
of osteocaicin to identify candidate compounds that will activate or inhibit
osteocalcin activity. The invention also includes compounds identified using
the methods of the invention. The invention also includes derivatives of
osteocalcin and bisphosphonate and tetracycline derivatives that act to
inhibit osteocalcin-hydroxyapatite binding. Furthermore, the invention relates
to the use of these compounds/derivatives in the treatment of diseases or
degenerative conditions resulting from increased or decreased bone
resorptionlformation and bony metastases of cancers, such as bone
metabolic disorders, osteoporosis, breast cancer, prostate cancer, lung
cancer and hypercalcemia malignancy.
Background of the Invention
All bone types consist of mineralized collagen fibrils as their building
block.
Each fibril is a type I collagen which is made up of three polypeptide chains
about 1000 amino acids long. The chains are wound together forming a
triple helix. The average diameter of a triple helix is about 1.5 nm and
length
of 300 nm. In bone, the fibrils are embedded with hydroxyapatite ("HA")
crystals (Ca~o(P04)6(OH)2). These crystals also contain carbonate,
magnesium, fluoride, and other impurities [2]. The bone crystals are plate-
shaped with average dimensions of 50 x 25 x 1.5-4.0 nm [1].
Bone undergoes constant turnover. The turnover process involves the break
down of bone by osteoclasts {specialized cells that break down bone) and
simultaneous rebuilding by osteoblasts (specialized cells that lay down new
bone). This process occurs at discrete sites named basic multicellular units
(BMUs), which contain the activities of both osteaclasts and osteoblasts,
though in different regions of the BMU [3]. During the turnover process, a
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CA 02446527 2003-10-23
number of extracellular proteins are produced. Some of the major bone
matrix proteins include type I collagen, proteoglycans, bone sialoprotein,
bone morphogenic proteins, osteonectin, osteopontin, and osteocalcin.
With the exception of collagen and bone morphogenic proteins, which
provide tensile strength and promote differentiation of bone cells
respectively, the functions of these proteins are still speculative.
Osteocalcin
is closely linked to the process of bone mineralization and bone turnover.
Many bone disorders in humans and other mammals are associated with
abnormal bone turnover. Such disorders include, but are not restricted to,
osteoporosis, Paget's disease, periodontal disease, tooth loss, bone
fractures, rheumatoid arthritis, periprosthetic osteolysis, osteogenesis
imperfecta, metastatic bone disease, hypercalcemia of malignancy, and
multiple myeloma. The most common of these disorders is osteoporosis.
Osteoporosis is a skeletal disease characterized by a low bone mass and
microarchitectural deterioration of bone tissue, with a consequent increase
in bone fragility and susceptibility to fracture. Up to 20% of women over 50
year of age have osteoporosis. Furthermore, bony metastases of cancers,
including breast, prostate and lung cancers, cause intolerable pain leading
to death. Up to 70% of breast and prostate cancer deaths consisted of bony
metastases. There is currently no cure or successful treatment for cancer
metastases to bone. There is a significant need to both prevent and treat
osteoporosis and bony cancer metastases as well as other conditions
associated with bone metabolism.
Osteocalcin is the most abundant non-collagenous protein found
associated with the mineralized bone matrix and it is currently being used
as a biological marker for clinical assessment of bone turnover. Osteocalcin
is a small (46-50 residue) bone specific protein that contains 3 gamma-
carboxylated glutamic acid residues in its primary structure. The name
osteocalcin (osteo, Greek for bone; Calc, Latin for lime salts; in, protein)
derives from the protein's ability to bind Ca2+ [4] and its abundance in bone
[5-7]. Osteocalcin is also known as "bone gamma-carboxyglutamic acid
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protein" (BGP) and "vitamin K-dependent protein of bone". It is distinguished
by the presence of 3 gamma-carboxylated giutamic acids (Gla), although
some human osteocalcin has been shown to contain only 2 Gla residues
(8]. Because the primary sequence of osteocalcin is highly conserved
among species (Figure 1) and it is one of the ten most abundant proteins in
the human body [9], it is reasonable to infer that the function of osteocalcin
is important.
The primary structure of osteocalcin from all species share extensive
identity (see table 1), suggesting that its function is preserved throughout
evolution. Conserved features include 3 Gla residues at positions 17, 21,
and 24, a disulfide bridge between Cys23 and Cys29, and most species
contain a hydroxyproline at position 9. The N-terminus of osteocalcin shows
highest sequence variation in comparison to other parts of the molecule.
Conformational study of osteocalcin by circular dichroism (CD) has shown
the existence of alpha-helical conformation in osteocalcin and that addition
of Ca2+ induces higher helical content [4, 10]. Two-dimensional nuclear
magnetic resonance (NMR) studies of osteocalcin in solution, while
structurally inconclusive, revealed that the calcium-free protein was
effectively unstructured except for the turn required by the disulfide bridge
between Cys23 and Cys29. All the proline residues (Hyp9, Pro11, Pro13,
Pro15, and Pro27) were in the traps conformation. Beta-turns are present in
the region of Tyr12, Asp14 and Asn26. The hydrophabic core of the molecule
is composed of the side chains of Leu2, Leu32, Va136 and Tyr42. The
calcium-induced helix is extremely rigid due to, in part, the hydrophobic
stabilization of the helical domain by the C-terminal domain [10].
Osteocalcin in solution binds Ca2+ with a dissociation constant ranging from
0.5 to 3 mM, with a stoichiometry of between 2 and 5 mol Ca2*/mol protein
[4, 11 ]. It has been suggested that osteocalcin binds to hydroxyapatite (Kd
10-' M) [8]. It appears that the Gla residues in osteocalcin are important for
its affinity toward Ca2+. Binding of Ca2+ induces normal osteocalcin to adopt
the alpha-helical conformation; however, thermally decarboxylated
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osteocalcin showed higher alpha-helical content than normal osteocalcin
and the calcium induced alpha-helical formation is lost [4]. Decarboxylated
osteocalcin also lost its specific binding to hydroxyapatite [8, 12]. When
bound to hydroxyapatite, the Gla residues are protected from thermal
decarboxylation [8]. Furthermore, osteocalcin synthesized in animals treated
with warfarin, which inhibits the formation of Gla, failed to bind to bone [13-
15]. Fourier-transform infrared (FT-IR) spectroscopic studies have shown
that the Gla residues in osteocalcin coordinate to Ca2+ in the malonate
chelation mode, where a Ca2~ interacts with two oxygen atoms, one from
each of the two COO- groups of a single Gla residue [16]. The binding
affinity of osteocalcin for hydroxyapatite increased fivefold by the addition
of 5
mM Ca2+ [4]. Furthermore, hydroxyapatite competition studies demonstrated
that prothrombin (10 Glalmolecule) and decarboxylated osteocalcin fail to
compete with X251-labeled osteocalcin bound to hydroxyapatite [12].
Combining all the information discussed above, a structural model has
been constructed [12]. This model consists of two antiparallel alpha-helical
domains. The Gla residues are spaced about 5.4 A apart on one of the
helices, which is similar to the interatomic lattice spacing of Ca2+ in the x~
plane of hydroxyapatite. It was therefore, predicted that the Gla residues in
osteocalcin bind to the (001) plane of hydroxyapatite lattice [4, 12].
In addition to osteocalcin's affinity to hydroxyapatite, it has also been
shown
that the transition of brushite (CaHP04.2H20) to hydroxyapatite
(Ca,o(P04)6(OH)2) is inhibited by very iow concentrations of osteocalcin [12].
The first in vivo indication of osteocalcin involvement with the
mineralization
of bone was demonstrated by Hauschka et. al. that osteocalcin appears in
embryonic chick bones coincident with the onset of mineralization [13].
Studies of bone physiology in animals maintained on warfarin, which
inhibits vitamin K-carboxylase, further supports the importance of
osteocalcin in bone mineralizatian. Rats maintained on warfarin during 8
months showed a dramatic closure of the epiphyseal growth plate, causing
a cessation of the longitudinal growth [17]. Lambs that were maintained on
high doses of warfarin from birth to 3 months of age had a significant
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decrease of trabecular bone turnover, a decrease of bone resorption, and a
dramatic reduction of the bone formation rate [15]. These animal studies
suggest that osteocalcin, possibly along with other Gfa-containing proteins,
is important for bone turnover. Ducy et. al. demonstrated that mineralized
bone from osteocalcin-deficient mice was two times thicker than that of wild-
type. It was shown that the absence of osteocalcin led to an increase in
bone formation without impairing bone resorption and did not affect
mineralization [18J. Ducy et. al. further suggested that osteocalcin may bind
to a specific, yet to be identified, receptor to fulfil its function. As a
consequence of this suggestion, Bodine et. al. demonstrated that
conditionally immortalized human osteoblasts metabolically responded to
osteocalcin in solution. Pretreatment of cells with inhibitors of adenylyl
cyclase, phopholipase C, and intracellular calcium release inhibited the
response of the cells to osteocalcin. It was concluded that these results
indicated that osteoblasts express an osteocalcin receptor, and this putative
receptor is coupled to a G-protein [19]. Evidence for the existence of an
osteocalcin receptor on osteoclasts has also been demonstrated.
Osteocafcin has been shown to induce chemotaxis, cellular differentiation,
and calcium-mediated intracellular signaling in osteoclast-like cells, derived
from giant cell tumors of bone [20].
Information about the functional role of osteocalcin is fragmented and
sometimes contradictory, the precise function of osteocalcin and
mechanism of action are yet elusive. The mechanism of osteocalcin's
action has been difficult to elucidate due to, in part, the tact that it has
no
known enzymatic activities. Its activity is apparently conferred only by
physical interactions with its target(s), which is undoubtedly dependent on
the structural characteristics of osteocalcin. Therefore, a detailed 3D
structure of osteocalcin is essential for the understanding of its function
and
such structure would be of great use in the design and screening for specific
modulators, activators or inhibitors of osteocaicin activity. Crystallization
of
osteocalcin from a fish has previously been reported (Coelho et al.
"Crystallization of Osteocalcin from a Marine Fish, Argyrosomus regius." 9th
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CA 02446527 2003-10-23
International Conference on the Crystallization of Biological
Macromolecules, March 23-28, 2003, Jena, Germany.) To date, osteocalcin
has not been crystallized in mammals. StructuraB determination of small
proteins is rather difficult because (i) heavy atom derivatives tend to
destroy
the crystal and (ii) another method that involves formation of seleno-
methionine, which is commonly used, cannot be used here because the
host that makes seleno-protein (E. coli) cannot make Gla. Therefore, in the
case of osteocalcin, having the protein crystallized does not automatically
lead to its 3-dimensional structure. Therefore, the reported crystalline form
from fish does not equate to 3-dimensional structure. Furthermore, as will
be demonstrated by this invention, the crystal structure of porcine
osteocalcin revealed that the N-terminus is rather flexible. Figure 1 shows
that mammalian osteocalcins have a longer N-terminus, which therefore
makes them more difficult to crystallize.
Summary of the Invention
The present invention relates to the crystalline form of osteocalcin. The 3D
structure provides a detailed description of osteocalcin's active site and a
simple model for its binding to hydroxyapatite. A striking feature of the
structure is the ordered arrangement of calcium atoms in the dimer
interface. The arrangement of negatively charged residues on the H 1
surface is precise for calcium coordination in that the coordination
geometries are near perfect. Projection of conserved residues onto the
molecular surface of the porcine osteocalcin structure reveals a striking and
extensive negatively charged surface centering on helix H1. By docking this
surface to the surface of hydroxyapatite, it was shown that the Gla surface of
osteocalcin complemented well with the surface of hydroxyapatite.
Additionally, when osteocalcin is bound to the surFace of hydroxyapatite,
other regions including the C-terminus of the protein, which has been
shown to possess chemotactic activity [29], would be well oriented to carry
out recruitment and signal transduction functions via binding to cell surface
receptors) on osteoclasts and osteoblasts. Accordingly, the invention
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includes a crystal comprising Osteocalcin, of resalution not less than 1.5
Angstroms. The crystalline osteocalcin preferably has at least one of the
following: (i) a conserved surface with a crystal structure which is created
by
atoms from 5 or less metal ions and from the following amino acid
residues: GIa17, GIa21, GIa24, Asp30 and Asp34; (ii) a structure comprising
three helices, most preferably connected by turns; (iii) a disulfide bridge
between Cys23 and Cys29; (iv) Ca2+, wherein osteocalcin comprises
amino acid residues GIa17, G(a21, GIa24, Asp30 and Asp34, and wherein
GIa17, GIa21, GIa24 and Asp30 coordinate the Ca2+.
Now that the three-dimensional structure of the osteocalcin crystal has been
determined, an inhibitorlmodulator of osteocalcin-hydroxyapatite binding
can be identified through the use of rational drug design by computer
modeling with a docking program. This procedure can include computer
fitting of potential inhibitors to the osteocalcin-hydroxyapatite binding to
ascertain how well the shape and the chemical structure of the potential
modulator will bind to hydroxyapatite to compete out osteocalcin. Computer
programs can also be employed to estimate the attraction, repulsion, and
steric hindrance of the subunits with a modulator/inhibitor. A particular
advantage is that selective inhibitors can be identified by comparing the
potential inhibitor to the 3D structure of osteocalcin.
The invention includes an isolated and purified molecule comprising a
binding pocket of osteocalcin defined by the structural coordinates of amino
acid residues GIa17, GIa21, Gta24, Asp30 and Asp34 according to Table 3
and/or other binding pocket amino acids described in this application. The
invention also includes an isolated and purified polypeptide consisting of a
portion of osteocalcin starting amino acid Pro13 and ending at one of amino
acids Asn27 to Tyr 46 of osteocalcin as set forth in the pig sequence shown
in Fig. 1. Other fragments of osteocaicin and corresponding amino acid nos
in other osteocalcins are also included within the scope of the invention.
The invention also includes an isolated and purified protein having the
structure defined by the structural coordinates shown in Table 3. The
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invention also includes a computer model of osteocalcin generated with the
structural coordinates listed in Table 3. Accordingly, the invention also
includes a method of identifying a compound that modulates (i.e. increases
or decreases) osteocalcin activity, comprising obtaining a 3D structure of
osteocalcin or fragment thereof, designing a compound to interact with, or
mimic, the 3D structure of osteocalcin or fragment thereof, obtaining the
compound, and determining whether the compound affects osteocalcin
activity. Mimicing the 3D structure of osteocalcin refers to providing a 3D
structure that is similar enough in 3D structure to osteocalcin that it is
able
to bind hydroxyapatite. A compound that mimics the 3D structure of
osteocalcin may be a competitive inhibitor of osteocalcin binding to
hydroxyapatite. The designing may be by comparison of a known
compound structure or by design (assembly) of a new or known compound
structure. The design of the compound preferably interacts with the
conserved surface of the osteocalcin or fragment thereof that binds to the
hydroxyapatite crystal.
The 3D structure preferably has at least one of the following: (i) a conserved
surface with a crystal structure which is created by atoms from 5 or less
metal ions and from the following amino acid residues: Gia17, Gfa21,
GIa24, Asp30 and Asp34; (ii) a structure comprising three helices, most
preferably connected by turns; (iii) a disulfide bridge between Cys23 and
Cys29; (iv) Ca2+, wherein osteocalcin comprises amino acid residues
G1a17, GIa21, GIa24, Asp30 and Asp34, and wherein GIal7, GIa21, Gfa24
and Asp30 coordinate the Ca2+.
The method optionally further comprises determining whether the
compound interacts with the hydroxyapatite and inhibits osteocalcin activity.
The method preferably further comprises: obtaining or synthesizing the
compound, forming hydroxyapatite:compound complex and analysing the
complex by X-ray crystallography to determine the ability of the compound to
interact with hydroxyapatite.
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The method also optionally further comprises:
a) determining the three-dimensional structure of the supplemental crystal
with molecular replacement analysis;
b) identifying or designing an inhibitor by performing rational drug design
with the three-dimensional structure determined for the supplemental
crystal or a fragment thereof.
The invention includes a compound obtained according to a method of the
invention.
The invention also includes the use of osteocalcin derivatives to interfere
with osteocalcin binding to hydroxyapatite. Such derivatives retain the
features on the hydroxyapatite binding surface but change the features on
the remaining surfaces such that osteocalcin will not interact with
cells/proteins in the original manner. Examples of such derivatives include:
(i) osteocalcin purified from non-human species, including but not limiting to
pig, monkey, cow, sheep, goat, dog, cat, rabbit, wallaby, rat, mouse,
xenopus, emu, chicken, carp, tetraodon, fugu, bluegill, seabream,
swordfish, other fish species, bird species, non-vertebrates
ii) mutating residues in 1-17 andJor 25-end
iii) insertion of residues into 1-17 and/or 25-end
iv) deletion of residues from 1-17 and/or 25-end
v) chemical modification of residues in 1-17 andlor 25-end using standing
chemical modification techniques.
In another embodiment, only the features on the HA binding surface are
altered. This will direct the osteocalcin derivatives to a location other than
bone and allow it to compete with the bone-bound osteocalcin in interacting
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with cellular protein and reduce the recruitment of cell to bone. Such
osteocalcin derivative may include, but are not limited to
i) de-carboxylation of Glas by chemical means or by expressing osteocalcin
in host cells that cannot synthesize GLA.
ii) Mutation of residues on the surface, as can be deduced from the 3-D
structure (including but not limiting to Gla-17, Gla-21, Gla-24, Asp-30, Asp-
34), that can cause steric clash with the hydroxyapatite surface and therefore
prevent the OCN mutant from interacting with the HA surface.
In a further embodiment, bisphosphonate derivatives and tetracycline
derivatives may be used to bind the hydroxyapatite surface, thereby inhibiting
osteocalcin binding.
The invention also includes a method for identifying a cornpoundlderivative
that inhibits osteocalcin activity, comprising:obtaining osteocalcin with
recombinant technology, chemical synthesis or purification from bone;
contacting osteocalcin with hydroxyapatite; subsequently adding a test
compound to compete with the bound osteocalcin for hydroxyapatite and
measuring the amount of osteocalcin dissociated from hydroxyapatite as a
result of the addition of the test compound; whereby a compound that
competes with osteocalcin for hydroxyapatite is identified as a compound
that inhibits osteocalcin activity.
Another aspect of the invention includes a method of treating a disease or
degenerative condition in a subject, comprising administering to the subject
a compound/derivative of the invention or a compound identified with a
method of the invention. The diseases or degenerative conditions include
those that result from increased or decreased bone resorptionlformation
and bony metastases of cancers, such as bone metabolic disorders,
osteoporosis, breast cancer, prostate cancer, lung cancer and
hypercalcemia malignancy.
CA 02446527 2003-10-23
The invention also incudes methods fo treating bone disease in a subject
comprising administering an effective amount of warfarin, aspirin or
deriviatives of either of the foregoing to the subject.
The invention also includes a method for identifying a compound that
inhibits osteocalcin activity, comprising
providing osteocalcin,
contacting osteocalciin with hydroxyapatite,
adding a test compound to the osteocalciin and hydroxyapatite and
determining whether the test compound competes with the bound
osteocalcin for hydroxyapatite by measuring the amount of
osteocalcin dissociated from hydroxyapatite as a result of the addition
of the test compound, wherein a compound that competes with
osteocalcin for hydroxyapatite is identified as a compound that
inhibits osteocalcin activity.
In the method, the test compound optionally includes fragments of
osteocalcin, such as fragments containing GIa17, GIa21 and GIa24 or
fragments containing Pro13 to Tyr46 or Pro 13 to Asn27. The osteocalcin is
optionally produced by chemical synthesis or recombinant methods and
may be produced as a modified osteocalcin molecule. For example, the
modified osteocalcin may lack the gamma-carboxylic acids on residues
GIa17, GIa31 and GIa24. Test compounds include bisphosphonates and
tetracycline as well as a derivative of either of the foregoing.
Brief Description of Drawings
Preferred embodiments are described in relation to the drawings, in which:
Figure 1. Sequence alignment of Osteocalcin. Protein sequence with the
secondary structure elements indicated and the conserved residues
highlighted (green, red, blue, yellow, orange and grey indicate conserved,
acidic, basic, cysteine, asparagine and glycine residues, respectively).
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Positions are identified as conserved if more than 85% of the residues are
identical, or similar if hydrophobic in nature. 'y' indicates a Gla residue,
open
triangles and circles indicate hydrophobic core and Ca2+-coordinating
surface, respectively.
Figure 2. Crystal structure of porcine osteocalcin and the experimental
electron density map. Porcine osteocalcin is shown as rod bond model,
where carbon, oxygen, nitrogen and sulfur atoms are coloured grey, red,
blue, and orange, respectively. The solvent-flattened SAS map (contoured at
1.5 sigma) is shown as green mesh. Calcium ions are shown as purple
spheres.
Figure 3. Structure of porcine osteocalcin. a, Protein sequence with the
secondary structure elements indicated and the conserved residues
highlighted (green, red, blue, yellow, orange and grey indicate conserved,
acidic, basic, cysteine, asparagine and glycine residues, respectively).
Positions are identified as conserved if more than 85% of the residues are
identical, or similar if hydrophobic in nature (see Supplementary Information
for the full sequence alignment). 'y' indicates a Gla residue, open triangles
and circles indicate hydrophobic core and Cap+-coordinating surface,
respectively. b, Ribbon representation of the crystal structure. The N and C
termini are labelled. Side chains of the Caz+coordinating residues and
those involved in tertiary structure stabilization are shown in stick
representation. Broken grey line indicates a hydrogen bond. c, d, Molecular
surface representations of porcine osteocalcin with the surface hydrophobic
patch (green) and the Ca2+coordinating surface (red) highlighted. Views in b
and c are perpendicular to that in d. e, Crystallographic dimer interface.
Orange and blue distinguish the two molecules. Purple spheres and the
yellow broken lines represent Caz+ ions and ionic bonds, respectively.
Figure 4. Model of porcine osteocalcin engaging an hydroxyapatite crystal
based on a Ca2+ ion lattice match. Only the best search solution is shown.
a, Alignment of porcine osteocalcin-bound (purple) and hydroxyapatite
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(green) Ca2+ ions. b, c, Orientation of porcine osteocalcin-bound Ca2+ ions
in a sphere of hydroxyapatite-Ca lattice (b) and on the hydroxyapatite
surface (c). In b, the parallelogram indicates a unit cell; the box
approximates the boundary of the slab shown in c and d. d, Docking of
porcine osteocalcin (orange backbone with grey semitransparent surface)
on hydroxyapatite. e, Detailed view of d showing the Ca-O coordination
network at the porcine osteocalcin-hydroxyapatite interface. Yellow broken
lines denote ionic bonds. Isolated red spheres and the tetrahedral clusters
of magenta and red spheres represent OH- and P04-3 ions, respectively.
Figure 5. Comparison of the top four solutions in the calcium lattice match
search. R.m.s.d. in distance between the porcine osteocalcin-bound and
the hydroxyapatite calcium ions are 0.44 A°, 0.47 A°, 0.61
A° and 0.61 A° in
(i)-(iv). a-d, Refer to legend in figure 4a-d for the corresponding
explanations.
Figure 6. a, Sedimentation equilibrium analyses in the presence of 10 mM
CaCl2. The best fit is shown as a line through the experimental points, and
the corresponding distributions of the residuals are shown above the plots.
b, Sedimentation equilibrium analyses in the absence of Calcium. The best
fit is shown as a line through the experimental points, and the
corresponding distributions of the residuals are shown above the plots.
Detailed Description of the Invention
Porcine osteocalcin crystallized as a crystallographic dimer with a two fold
symmetry about the b-axes. There is no direct intermolecular protein-protein
interaction within the dimer, but rather, the interactions that hold the dimer
together are protein-Ca2+-protein. The Gla residues on each monomer are
arranged linearly on the protein surface and the row of Ca2+ is sandwiched
between these two surfaces. The arrangement of the Ca2+ atoms is ordered
and also has the same 2-fold relationship. The crystal structure POC~3~9
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CA 02446527 2003-10-23
consists of 3 helices, separated another by forming
each is from a turn a
helix-turn-helix-turn-helixThe first (H1) spans Asp17
motif. helix from to
Asn26. All three Gla lie on one of H1 helix their
residues side with side-
chains radiating away from the protein core where they, together with Asp30,
coordinate the 5 Ca2+ ions that form the dimer interface. The second helix
(H2) spans from Asp28 to Asp34. H2 is separated from H1 by a turn
between Leu25 and Pro27, which is stabilized by the disulfide-bridge. The
turn and the disulfide-bridge position H2 such that Asp30 is oriented
correctly for participation in calcium chelation. The third helix (H3) spans
from Phe38 to Tyr46. H3 is turned back, via a turn between I1e36 and Phe38,
to close proximity of H1 and H2; thereby, forming a hydrophobic core
between the three helixes. The hydrophobic core is made up of residues
Va122, Leu25, Leu32, A1a33, A1a41, Tyr42, Phe45 and Tyr46.
A striking feature of the structure is the ordered arrangement of calcium
atoms in the dimer interface. Such order is characteristic of crystal
lattices,
showing that the calcium binding surface on osteocalcin is also suited for
binding to crystal surfaces. The arrangement of negatively charged residues
on the H1 surface is precise for calcium coordination in that the coordination
geometries are near perfect. Projection of conserved residues onto the
molecular surface of the porcine osteocalcin structure reveals a striking and
extensive negatively charged surface centering on helix H1. By docking this
surface to the surface of hydroxyapatite we found that the Gla surface of
osteocalcin complemented well with the surface of hydroxyapatite.
Additionally, when osteocalcin is bound to the surface of hydroxyapatite,
other regions including the C-terminus of the protein, which has been
shown to possess chemotactic activity [30], would be well oriented to carry
out recruitment and signal transduction functions via binding to cell surface
receptors) on osteoclasts and osteoblasts.
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In one aspect the invention is directed to the three-dimensional structure of
an isolated and purified osteocalcin and its structure coordinates.
The invention also includes methods of identifying compounds capable of
inhibiting osteocalcin binding to hydroxyapatite.
Another aspect of the invention is to use the structural coordinates of
osteocalcin to homology model other osteocalcin-like species.
This invention provides the first rational drug design strategy for modulating
osteocalcin activity. The structure coordinates and atomic details of
osteocalcin are useful to design, evaluate (preferably computationally) and
synthesize inhibitors of osteocaicin that prevent or treat bone pathologies.
The invention includes methods for identifying compounds that can interact
with osteocalcin or the binding site for osteocalcin on hydroxyapafiite: The
method for identifying inhibitors preferably include fitting structures of
osteocalcin domains into the 3D sfiructure of the inhibitor bound osteocalcin.
These interactions can be easily identified by comparing the structural,
chemical and spatial characteristics of a candidate compound to the three
dimensional structure of the osteocalcin. Since the amino acids that are
responsible for osteocalcin activity and binding were identified by this
invention, drug design may be done on a rational basis.
The structure series as a detailed basis for the design and testing of
inhibitors, initially in the computer, but also in vitro in cell culture and
in vivo,
prouding a method for identifying inhibitors having specific contacts with the
osteocalcin or an isoform, homologue or mutant or the osteocalcin binding
site on hydroxyapatite. The effect of a modification to a substrate or
inhibitor
may be readily viewed on a computer, without the need to synthesize the
compound and assay it in vitro. As well, non-protein organic molecules may
also be compared to the osteocalcin on a computer. One can readily
determine if the molecules have suitable structural and chemical
characteristics to interact with, or activate or inhibit, osteocalcin
activity. The
invention includes the osteocalcin modulators discovered using all or part of
CA 02446527 2003-10-23
an osteocalcin of the invention (preferably the 3D structure) and the
methods of the invention.
16
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CI"~/Sta~S
Crystal Properties
The crystal structure of porcine osteocalcin was determined at 2.0
A° using
the Iterative Single Anomalous Scattering method [10p]. Bijvoet difference
Patterson map analysis detected the presence of three tightly bound Ca2+
ions and two S atoms corresponding to a disulphide bridge between Cys 23
and Cys 29, which together were used to phase the porcine osteocalcin
structure. An atomic model corresponding to residues Pro 13 to Ala 49 was
built into well-defined electron density (Figure 2) and refined to an RWOrk
and
R~.ee of 25.5% and 28.3%, respectively. Data collection and structure
refinement statistics are summarized in Table 2.
Porcine osteocalcin forms a tight globular structure comprising a previously
unknown fold (no matches in the DALI database11 ) with a topology
consisting, from its amino terminus, of three a-helices (denoted a1-a3)
and a short extended strand (denoted Ex1; Figure 3b). Helix a1 and helix a2
are connected by a type III turn structure from Asn 26 to Cys 29 and form a V
shaped arrangement that is stabilized by an interhelix disulphide bridge
involving Cys 23 and Cys 29. Helix a3 is connected to helix a2 by a short
turn and is aligned to bisect the V-shape arrangement of helix a1 and helix
a2. The three a-helices together compose a tightly packed core involving
conserved hydrophobic residues Leu 16, Leu 32, Phe 38, Ala 41, Tyr 42,
Phe 45 and Tyr 46. The overall tertiary structure is further stabilized by a
hydrogen bond interaction between two invariant residues, Asn 26 in the
helix a1-a2 linker and Tyr 46 in helix a3.
Projection of conserved residues onto the molecular surface of the pOC
structure (Figure 3c, d) shows an extensive negatively charged surface
centring on helix a1 (solvent-exposed surface area 586 A°2). Notably,
all
three Gla residues implicated in hydroxyapatite binding are located on the
same surface of helix a1 and, together with the conserved residue Asp 30
from helixa2, coordinate five Ca2+ ions (denoted Ca1-Ca5) in an elaborate
17
CA 02446527 2003-10-23
network of ionic bonds (Figure 3e). These five Ca2+ ions are sandwiched
between two crystallographically related porcine osteocalcin molecules and
show both monodentate and malonate modes of chelation with extensive
bridging.
In the porcine osteocalcin crystal structure, the Ca2+ ions coordinated by the
Gla residues have an unexpected periodic order reminiscent of a crystalline
lattice. Because Gla residues are essential for the interaction of osteocalcin
with bone in vivo (34] and for the specific interaction with hydroxyapatite in
vitro [31], whether the specific atomic arrangement of bound Ca2+ ions in
the pOC crystal structure mimics the spatial arrangement of Ca2+ ions in
hydroxyapatite was investigated. To do so, a comprehensive real-space
search for a spatial match between the pOC-bound Ca2+ ions and the Ca2+
ions in crystalline hydroxyapatite (Ca5(P04)30H, space group P63/m, unit-cell
dimensions a = b = 9.432 A°, c = 6.881 A° ) [35] was done.
Search solutions
were ranked by root mean square (r.m.s.) deviations of distances between
osteocalcin bound and hydroxyapatite Ca2+ ions.
Unique solutions within 1 s.d. (0.29 A°) of the best solution were
chosen for
graphical analysis. Molecular surfaces of hydroxyapatite defined by the Ca2+
ions of the best search solutions were constructed for docking analysis
(Figure 4 and Figure 5). The best (r.m.s. deviation 0.44 A°) and fourth
best
(r.m.s. deviation 0.62 A°) solutions in the search correspond to the
prism
face (100) of HA, whereas the secand (r.m.s. deviation 0.47 A°) and
third
(r.m.s. deviation 0.61 A°) best solutions correspond to the secondary
prism
face (110). Notably, the prism face is the predominant crystal face
expressed in geological [36] and synthetic hydroxyapatite [37] and, although
the predominant crystal face of hydroxyapatite expressed in bone has not
been unambiguously determined, atomic force microscopy (38] and
diffraction analysis [39] indicate that the expressed face lies parallel to
the
crystal c axis. The best solution identified in the search also corresponds to
a crystal face that lies parallel to the crystal c axis.
Although the best and second best solutions both show good lattice match
18
CA 02446527 2003-10-23
statistics, only the best solution gives rise to a docking mode of porcine
osteocalcin to hydroxyapatite that is free of steric clash. Using the best
search solution, a more detailed binding model was generated. The
coordination network of Ca-O atoms at the osteocalcin-hydroxyapatite
interface closely mimics that in the hydraxyapatite crystal lattice (r.m.s.
deviation Ca-O bond distance, 0.19 A°; r.m.s. deviation Ca-O-Ca bond
angle, 9.63°; Figure 4e).
The hydroxyapatite lattice binding mode represented by the best solution
presupposes that osteocalcin engages hydroxyapatite with the acidic Ca2+-
coordinating surface as a monomer. In the crystal structure of porcine
osteocalcin, however, the five Ca2+ ions are sandwiched between two
crystallographically related protein molecules. If overly stable, this dimeric
state could present an impediment to hydroxyapatite binding. To investigate
whether porcine osteocalcin exists as a monomer or dimer in solution,
sedimentation equilibrium analysis in the presence and absence of 10mM
CaCl2 was carried out. In both cases, the sedimentation equilibrium data
were best fitted to a monomer-dimer equilibrium model (Figure 6). The
extrapolated dissociation constants (Kd) for the osteocalcin dimer were 8 x
10-4 M and 2 x 10~ M in the absence and presence of 10mM CaGl2,
respectively. Theses Kd values are 2-3 orders of magnitude higher than the
concentration of osteocalcin in human serum (0.9 x 106 to 7 x 10-6 M) [30],
showing that osteocalcin exists as a monomer in vivo.
The crystal structure of porcine osteocalcin provides a first glimpse of the
underlying interactions that may constitute biomineral recognition. The
recognition of crystal lattices by proteins is important in many biological
processes, including the inhibition of ice crystal growth and the
development of teeth, bone and shells. The best-characterized
protein-crystal recognition system studied so far corresponds to the
interaction of antifreeze proteins (AFP) with ice [40, 41]. AFPs bind to the
surface of ice to modify crystal morphology and to inhibit ice growth. AFPs
with different three-dimensional structures bind to different planes of ice,
19
CA 02446527 2003-10-23
and the shape complementarity between the ice-binding surface of AFP and
the ice crystal surface to which it binds is the primary determinant for
binding specificity.
The excellent surface complementarity between the Ca2+-coordinating
surface of porcine osteocalcin and the prism face of hydroxyapatite shows
that porcine osteocalcin will also show selective binding characteristics to
hydroxyapatite. By analogy to the antifreeze proteins, the binding of
osteocalcin to hydroxyapatite could directly modulate hydroxyapatite crystal
morphology and growth. In addition, when osteocalcin is bound to the
surtace of hydroxyapatite, other regions, including the carboxy terminus of
the protein, which possesses chemotactic activity [42], would be we(I
orientated to carry out recruitment and signal transduction functions through
binding to cell surface receptors on osteoclasts [32] and osteoblasts (33].
The structure of porcine osteocalcin provides a model for further functional
analyses.
Three-Dimensional Configurations
X-ray structure coordinates define a unique configuration of points in space.
Those of skill in the art understand that a set of structure coordinates for
protein or an protein/ligand complex, or a portion thereof, define a relative
set of points that, in turn, define a configuration in three dimensions. A
similar or identical configuration can be defined by an entirely different set
of
coordinates, provided the relative distances and angles between
coordinates remain essentially the same.
The configurations of points in space derived from structure coordinates
according to the invention can be visualized as, for example, a holographic
image, a stereodiagram, a model or a computer-displayed image, and the
invention thus includes such images, diagrams or models.
Structurally Equivalent Crystal Structures
CA 02446527 2003-10-23
Various computational analyses can be used to determine whether a
molecule or the active site portion thereof is "structurally equivalent,"
defined
in terms of its three-dimensional structure, to all or part of osteocalcin or
its
binding sites. Such analyses may be carried out in current software
applications, such as the Molecular Similarity application of QUANTA
(Molecular Simulations Inc., San Diego, Calif.) version 4..1, and as described
in the accompanying User's Guide.
The Molecular Similarity application permits comparisons between different
structures, different conformations of the same structure, and different parts
of the same structure. The procedure used in Molecular Similarity to
compare structures is divided into four steps: (1) load the structures to be
compared; (2) define the atom equivalences in these structures; (3) perform
a fitting operation; and (4) analyze the results.
Each structure is identified by a name. One structure is identified as the
target (i.e., the fixed structure); all remaining structures are working
structures (i.e., moving structures). Since atom equivalency within QUANTA
is defined by user input, for the purpose of this invention equivalent atoms
are defined as protein backbone atoms (N, C.alpha., C, and O) for all
conserved residues between the two structures being compared. A
conserved residue is defined as a residue that is structurally or functionally
equivalent. Only rigid fitting operations are considered.
When a rigid fitting method is used, the working structure is translated and
rotated to obtain an optimum fit with the target structure. The fitting
operation
uses an algorithm that computes the optimum translation and rotation to be
applied to the moving structure, such that the root mean square difference of
the fit over the specified pairs of equivalent atom is an absolute minimum.
This number, given in angstroms, is reported by QUANTA.
For the purpose of this invention, any molecule or molecular complex or
active site thereof, or any portion thereof, that has a root mean square
deviation of conserved residue backbone atoms (N, Cec, C, O) of less than
21
CA 02446527 2003-10-23
about 1.5 A, when superimposed on the relevant backbone atoms
described by the reference structure coordinates listed in the tables, i s
considered "structurally equivalent" to the reference molecule. That is to
say,
the crystal structures of those portions of the two molecules are
substantially identical, within acceptable error. Particularly preferred
structurally equivalent molecules or molecular complexes are those that are
defined by the entire set of structure coordinates listed in the tables,
plus/minus a root mean square deviation from the conserved backbone
atoms of those amino acids of not more than 1.5 A. More preferably, the root
mean square deviation is less than about 1.0 A or 0.5 A.
The term "root mean square deviation'° means the square root of
the
arithmetic mean of the squares of the deviations. It is a way to express the
deviation or variation from a trend or object. For purposes of this invention,
the "root mean square deviation" defines the variation in the backbone of a
protein from the backbone of osteocalcin or a binding site portion thereof, as
defined by the structure coordinates of osteocalcin described herein.
Structurally Homologous Molecules, Molecular Complexes, and Crystal
Structures
The structure coordinates can be used to aid in obtaining structural
information about another crystallized molecule or molecular complex. The
method of the invention allows determination of at least a portion of the
three-dimensional structure of molecules or molecular complexes which
contain one or more structural features that are similar to structural
features
of osteocalcin. These molecules are referred to herein as "structurally
homologous" to osteocalcin. Similar structural features can include, for
example, regions of amino acid identity, conserved active site or binding site
motifs, and similarly arranged secondary structural elements (e.g., a-
helices and a-sheets). Optionally, structural homology is determined by
aligning the residues of the two amino acid sequences to optimize the
number of identical amino acids along the lengths of their sequences; gaps
22
CA 02446527 2003-10-23
in either or both sequences are permitted in making the aligmnent in order
to optimize the number of identical amino acids, although the amino acids
in each sequence must nonetheless remain in their proper order.
Preferably, two amino acid sequences are compared using the Blastp
program, version 2Ø9, of the BLAST 2 search algorithm, as described by
[43], and available at http:/lwww.ncbi.nlm.nih.gov/gorflb12.html. Preferably,
the default values for all BLAST 2 search parameters are used. In the
comparison of two amino acid sequences using the BLAST search
algorithm, structural similarity is referred to as "identity." Preferably, a
structurally homologous molecule is a protein that has an amino acid
sequence sharing at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% sequence identity with a native or recombinant
amino acid sequence of osteocalcin. More preferably, a protein that is
structurally homologous to osteocalcin includes at least one contiguous
stretch of at least 25 or 50 amino acids that shares at least 80% amino acid
sequence identity with the analogous portion of the native or recombinant
osteocalcin. Methods for generating structural information about the
structurally homologous molecule or molecular complex are well-known
and include, for example, molecular replacement techniques.
Therefore, in another embodiment this invention provides a method of
utilizing molecular replacement to obtain structural information about a
molecule or molecular complex whose structure is unknown comprising the
steps of:
(a) crystallizing the molecule or molecular complex of unknown structure;
{b) generating an x-ray diffraction pattern from said crystallized molecule or
molecular complex; and
(c) applying at least a portion of the structure coordinates to the x-ray
diffraction pattern to generate a three-dimensional electron density map of
the molecule or molecular complex whose structure is unknown.
23
CA 02446527 2003-10-23
By using molecular replacement, all or part of the structure coordinates of
osteocalcin as provided by this invention can be used to determine the
structure of a crystallized molecule whose structure is unknown more
quickly and efficiently than attempting to determine such information ab
initio.
Molecular replacement provides an accurate estimation of the phases for an
unknown structure. Phases are a factor in equations used to solve crystal
structures that cannot be determined directly experimentally. Obtaining
accurate values for the phases, by methods other than molecular
replacement, is a time-consuming process that involves iterative cycles of
approximations and refinements and greatly hinders the solution of crystal
structures. However, when the crystal structure of a protein containing at
least a structurally homologous portion has been solved, the phases from
the known structure provide a satisfactory estimate of the phases for the
unknown structure.
Thus, this method involves generating a preliminary model of a molecule
whose structure coordinates are unknown, by orienting and positioning the
relevant portion of osteocalcin to the structure coordinates listed within the
unit cell of the crystal of the unknown molecule or molecular complex so as
2o best to account for the observed x-ray diffraction pattern of the crystal
of the
molecule whose structure is unknown. Phases can then be calculated from
this model and combined with the observed x-ray diffraction pattern
amplitudes to generate an electron density map of the structure whose
coordinates are unknown. This, in turn, can be subjected to any well-known
model building and structure refinement techniques to provide a final,
accurate structure of the unknown crystallized molecule or molecular
complex [44, 45].
Structural information about a portion of any crystallized molecule that is
sufficiently structurally homologous to a portion of osteocalcin can be
resolved by this method. In addition to a molecule that shares one or more
structural features with osteocalcin as described above, a molecule that has
24
CA 02446527 2003-10-23
similar bioactivity, such as the same hydroxapatite binding activity as
osteocalcin, may also be sufficiently structurally homologous to osteocalcin
to permit use of the structure coordinates of osteocalcin to solve its crystal
structure.
In a preferred embodiment, the method of molecular replacement is utilized
to obtain structural information about a molecule, wherein the molecule
comprises at least one osteocalcin fragment or homolog. A "fragment" of
osteocalcin is an osteocalcin molecule that has been truncated at the N-
terminus or the C-terminus, or both. In the context of the present invention,
a
"homolog" of osteocalcin is a protein that contains one or more amino acid
substitutions, deletions, additions, or rearrangements with respect to the
amino acid sequence of osteocalcin, but that, when folded into its native
conformation, exhibits or is reasonably expected to exhibit at least a portion
of the tertiary (three-dimensional) structure of osteocalcin. For example,
structurally homologous molecules can contain deletions or additions of
one or more contiguous or noncontiguous amino acids, such as a loop or a
domain. Structurally homologous molecules also include "modified"
osteocalcin molecules that have been chemically or enzymatically
derivatized at one or more constituent amino acid, including side chain
modifications, backbone modifications, and N- and C-terminal
modifications including acetylation, hydroxylation, methylation, amidation,
and the attachment of carbohydrate or lipid moieties, cofactors, and the like.
A heavy atom derivative of osteocalcin is also included as an osteocalcin.
The term "heavy atom derivative" refers to derivatives of osteocalcin
produced by chemically modifying a crystal of osteocalcin. In practice, a
crystal is soaked in a solution containing heavy metal atom salts, or
organometallic compounds, e.g., lead chloride, gold thiomalate, thiomersal
or uranyl acetate, which can diffuse through the crystal and bind to the
surface of the protein. The locations) of the bound heavy metal atoms) can
be determined by x-ray diffraction analysis of the soaked crystal. This
CA 02446527 2003-10-23
information, in turn, is used to generate the phase information used to
construct three-dimensional structure of the protein [46].
The structure coordinates of osteocalcin as provided by this invention are
particularly useful in solving the structure of osteocalcin mutants. Mutants
may be prepared, for example, by expression of osteocalcin cDNA
previously altered in its coding sequence by oligonucleotide-directed
mutagenesis. Mutants may also be generated by site-specific incorporation
of unnatural amino acids into osteocalcin proteins using the general
biosynthetic method of [47]. In this method, the codon encoding the amino
acid of interest in wild-type osteocalcin is replaced by a "blank" nonsense
codon, TAG, using oligonucleotide-directed mutagenesis. A suppressor
tRNA directed against this codon is then chemically aminoacylated in vitro
with the desired unnatural amino acid. The aminoacylated tRNA is then
added to an in vitro translation system to yield a mutant with the site-
specific
incorporated unnatural amino acid.
The structure coordinates of osteocalcin are also particularly useful to solve
the structure of crystals of osteocalcin mutants or osteocalcin co-complexed
with hydroxyapatite. This approach enables the determination of the optimal
sites for interaction, including candidate osteocalcin inhibitorslmodulators.
Potential sites for modification within the various binding site of the
molecule can also be identified. This information provides an additional tool
for determining the most efficient binding interactions. For example, high
resolution x-ray diffraction data collected from crystals exposed to different
types of solvent allows the determination of where each type of solvent
molecule resides. Small molecules that bind tightly to those sites can then
be designed and synthesized and tested for their osteocalcin inhibition
activity.
All of the complexes referred to above may be studied using well-known x
ray diffraction techniques and may be refined versus 1.5-3.0 A resolution x
ray data to an R value of about 0.20 or less using computer software, such
as CNS. This information may thus be used to optimize known osteocalcin
26
CA 02446527 2003-10-23
inhibitorslmodulators, and more importantly, to design new osteocalcin
inhibitorslmodulators.
The invention also includes the unique three-dimensional configuration
defined by a set of points defined by the structure coordinates for a molecule
structurally homologous to osteocalcin as determined using the method of
the present invention, structurally equivalent configurations, and magnetic
storage media comprising such set of structure coordinates.
Further, the invention includes structurally homologous molecules as
identified using the method of the invention.
Homology Modeling
Using homology modeling, a computer model of an osteocalcin homolog
can be built or refined without crystallizing the homoiog. First, a
preliminary
model of the osteocalcin homolog is created by sequence alignment with
osteocalcin, secondary structure prediction, the screening of structural
libraries, or any combination of those techniques. Computational software
may be used to carry out the sequence alignments and the secondary
structure predictions. Structural incoherences, e.g., structural fragments
around insertions and deletions, can be modeled by screening a structural
library for peptides of the desired length and with a suitable conformation.
For prediction of the side chain conformation, a side chain rotamer library
may be employed. Where the osteocalcin homolog has been crystallized,
the final homology model can be used to solve the crystal structure of the
homolog by molecular replacement, as described above. Next, the
preliminary model is subjected to energy minimization to yield an energy
minimized model. The energy minimized model may contain regions where
stereochemistry restraints are violated, in which case such regions are
remodeled to obtain a final homology model.
27
CA 02446527 2003-10-23
Drug Design of Inhibitors
Inhibitors
Inhibitors of osteocalcin provide a basis for diagnosis andlor treatment of
bone-related pathologies. "Pathology" includes a disease, a disorder
andlor an abnormal physical state caused by increased or decreased bone
resorptionlformation and bony metastases of cancers such as bone
metabolic disorders, osteoporosis, breast cancer, prostate cancer, lung
cancer and hypercalcemia malignancy. The structures are useful in the
design of inhibitors, which may be used as therapeutic or prophylactic
compounds for treating pathologies in which downregulation of osteocalcin-
hydroxapatite binding is beneficial. It will be apparent that methods using
osteocalcin described below may be readily adapted for use with a fragment
of osteocalcin or osteocalcin variant.
The characterization of the novel binding surface permits the design of
potent, highly selective inhibitors. ;ieveral approaches can be taken for the
use of the structure in the rational design of inhibitors. A computer-
assisted, manual examination of an inhibitor binding site structure may be
done.
Rational Drug Design
Computational techniques can be used to screen, identify, select and
design chemical entities capable of associating with osteocalcin or
structurally homologous molecules. Knowledge of the structure coordinates
for osteocalcin permits the design andlor identification of synthetic
compounds and/or other molecules which have a shape complementary to
the conformation of an osteocalcin binding site. In particular, computational
techniques can be used to identify or design chemical entities, such as
inhibitors, agonists and antagonists, that associate with an osteocalcin
binding site. Inhibitors may bind to or interfere with all or a portion of the
binding site of osteocalcin to hydroxyapatite, and can be competitive, non-
28
CA 02446527 2003-10-23
competitive, or uncompetitive inhibitors. Once identified and screened for
biological activity, these inhibitors/agonistslantagonists may be used
therapeutically or prophylactically to block osteocalcin activity. Structure-
activity data for analogs of ligands that bind to or interfere with
osteocalcin -
like binding sites can also be obtained computationally.
Accordingly, the invention includes a method of designing a compound that
inhibits osteocalcin activity, compri sing performing rational drug design
with
a 3D structure of osteocalcin or fragment thereof to design a compound that
interacts with the 3D structure of hydroxyapatite or fragment thereof and
inhibits osteocalcin activity.
The invention also includes a method of identifying whether a compound
inhibits osteocalcin activity, comprising performing rational drug design with
a 3D structure of osteocalcin or fragment thereof, the drug design
comprising i) comparing the 3D structure of the compound to the 3D
structure of osteocalcin or fragment thereof and ii) determining whether the
compound interacts with the 3D structure of hydroxyapatite and inhibits
osteocalcin activity.
The drug design is preferably performed in conjunction with computer
modeling comprising introducing into a computer program structural
coordinates defining an osteocalcin or fragment thereof, wherein the
program generates the 3D structure of the osteocalcin or fragment.
In the method, the compound that inhibits osteocalcin preferably has a
greater affinity for the binding of hydroxyapatite than does osteocalcin.
The term "osteocalcin-like binding site" refers to a portion of a molecule or
molecular complex whose shape is sufficiently similar to at least a portion
of the active site of osteocalcin as to be expected to bind hydroxyapatite. A
structurally equivalent active site is defined by a root mean square deviation
from the structure coordinates of the backbone atoms of the amino acids
29
CA 02446527 2003-10-23
that make up the active site in ostoecalcin of at most about 1.5 A. How this
calculation is obtained is described below.
Accordingly, the invention thus provides molecules or molecular complexes
comprising an osteocafcin binding site or an osteocalcin -like binding site,
as defined by the sets of structure coordinates described above.
The term "chemical entity," as used herein, refers to chemical compounds,
complexes of two or more chemical compounds, and fragments of such
compounds or complexes. Chemical entities that are determined to
associate with osteocalcin are potential drug candidates.
Data stored in a machine-readable storage medium that is capable of
displaying a graphical three-dimensional representation of the structure of
osteocalcin or a structurally homologous molecule, as identified herein, or
portions thereof may thus be advantageously used for drug discovery. The
structure coordinates of the chemical entity are used to generate a three-
dimensional image that can be computationally fit to the three-dimensional
image of osteocalcin or a structurally homologous molecule. The three-
dimensional molecular structure encoded by the data in the data storage
medium can then be computationally evaluated for its ability to associate
with hydroxyapatite. When the molecular structures encoded by the data are
displayed in a graphical three-dimensional representation on a computer
screen, the protein structure can also be visually inspected for potential
association with hydroxyapatite.
One embodiment of the method of drug design involves evaluating the
potential association of a known chemical entity with osteocalcin, a
structurally homologous molecule or with the binding-site of osteocalcin on
hydroxyapatite. The method of drug design thus includes computationally
evaluating the potential of a selected chemical entity to associate with any
of
the molecules or molecular complexes set forth above. This method
comprises the steps of: (a) employing computational means to perform a
fitting operation between the selected chemical entity and a binding site, or
CA 02446527 2003-10-23
a pocket nearby the substrate binding site, of the molecule or molecular
complex; and (b) analyzing the results of said fitting operation to quantify
the
association between the chemical entity and the active site.
In another embodiment, the method of drug design involves computer-
s assisted design of chemical entities that associate with osteocalcin, its
homologs, portions thereof, or with the binding site of osteocalcin on
hydroxyapatite. Chemical entities can be designed in a step-wise fashion,
one fragment at a time, or may be designed as a whole or "de novo."
To be a viable drug candidate, the chemical entity identified or designed
according to the method must be capable of structurally associating with at
least part of osteocalcin or osteocalcin binding sites on hydroxyapatite, and
must be able, sterically and energetically, to assume a conformation that
allows it to associate. Non-covalent molecular interactions important in this
association include hydrogen bonding, van der Waals interactions,
hydrophobic interactions, and electrostatic interactions. Conformational
considerations include the overall three-dimensional structure and
orientation of the chemical entity in relation to the active site, and the
spacing between various functional groups of an entity that directly interact
with the osteocalcin -like active site or homologs thereof.
Optionally, the potential binding of a chemical entity to an osteocalcin or
osteocalcin binding site on hydroxyapatite is analyzed using computer
modeling techniques prior to the actual synthesis and testing of the
chemical entity. If these computational experiments suggest insufficient
interaction and association, testing of the entity is obviated. However, if
computer modeling indicates a strong interaction, the molecule may then be
synthesized and tested for its ability to interfere with an osteocalcin
binding
to hydroxyapatite. Binding assays to determine if a compound actually binds
can also be performed and are well known in the art. Binding assays may
employ kinetic or thermodynamic methodology using a wide variety of
techniques including, but not limited to, microcalorimetry, circular
dichroism,
31
CA 02446527 2003-10-23
capillary zone electrophoresis, nuclear magnetic resonance spectroscopy,
fluorescence spectroscopy, and combinations thereof.
One skilled in the art may use one of several methods to screen chemical
entities or fragments for their ability to associate with an osteocalcin or
osteocalcin binding site on hydroxyapatite. This process may begin by visual
inspection on the computer screen based on the osteocalcin structure
coordinates or other coordinates which define a similar shape generated
from the machine-readable storage medium. Selected fragments or
chemical entities may then be positioned in a variety of orientations, or
docked, within the active site. Docking may be accomplished using software
such as QUANTA and SYBYL, followed by energy minimization and
molecular dynamics with standard molecular mechanics forcefields, such
as CHARMM and AMBER.
Specialized computer programs may also assist in the process of selecting
fragments or chemical entities. Examples include GRID [48]; available from
Oxford University, Oxford, UK); MCSS [49]; available from Molecular
Simulations, San Diego, Calif.); AUTODOCK [50]; available from Scripps
Research Institute, La ,lolla, Calif.); and DOCK [51]; available from
University
of California, San Francisco, Calif.).
Once suitable chemical entities or fragments have been selected, they can
be assembled into a single compound or complex. Assembly may be
preceded by visual inspection of the relationship of the fragments to each
other on the three-dimensional image displayed on a computer screen in
relation to the structure coordinates of osteocalcin. This could be followed
by manual model building using software such as QUANTA or SYBYL
(Tripos Associates, St. Louis, Mo.).
Useful programs to aid one of skill in the art in connecting the individual
chemical entities or fragments include, without limitation, CAVEAT (52, 53];
available from the University of California, Berkeley, Calif.); 3D database
systems such as ISIS (available from MDL Information Systems, San
32
CA 02446527 2003-10-23
Leandro, Calif.; reviewed in Y. C. Martin [54]; and HOOK [55]; available from
Molecular Simulations, San Diego, Calif.).
Osteocalcin or hydroxyapatite binding compounds may be designed "de
novo" using either an empty binding site or optionally including some
portions) of a known inhibitor(s). There are many de novo ligand design
methods including, without limitation, LUDI [56]; available from Molecular
Simulations Inc., San Diego, Calif.); LEGEND [57]; available from Molecular
Simulations Inc., San Diego, Calif.); LeapFrog (available from Tripos
Associates, St. Louis, Mo.); and SPROUT [58]; available from the University
of Leeds, UK).
Once a compound has been designed or selected by the above methods,
the efficiency with which that entity may bind to or interfere with an
osteocalcin or osteocalcin binding site on hydroxyapatite may be tested and
optimized by computational evaluation.
An entity designed or selected as binding to or interfering with an
osteocalcin may be further computationally optimized so that in its bound
state it would preferably lack repulsive electrostatic interaction with its
target
and with the surrounding water molecules. Such non-complementary
electrostatic interactions include repulsive charge-charge, dipole-dipole,
and charge-dipole interactions.
Specific computer software is available in the art to evaluate compound
deformation energy and electrostatic interactions. Examples of programs
designed for such uses include: Gaussian 94, revision C (M. J. Frisch,
Gaussian, Inc., Pittsburgh, Pa. 151 p6); AMBER, version 4.1 (P. A. Kollman,
University of California at San Francisco, 94143); QUANTA/CHARMM
(Molecular Simulations, Inc., San Diego, Calif. 92121); Insight IIIDiscover
(Molecular Simulations, Inc., San Diego, Calif. 92121 ); DeIPhi (Molecular
Simulations, Inc., San Diego, Calif. 92121 ); and AMSOL (Quantum
Chemistry Program Exchange, Indiana University). These programs may be
implemented, for instance, using a Silicon Graphics workstation such as an
33
CA 02446527 2003-10-23
Indigo2 with "IMPACT" graphics. Other hardware systems and software
packages will be known to those skilled in the art.
Another approach encompassed by this invention is the computational
screening of small molecule databases for chemical entities or compounds
that can bind in whole, or in part, l:o an osteocalcin or osteocalcin binding
site on hydroxyapatite. In this screening, the quality of fit of such entities
to
the binding site may be judged either by shape complementarity or by
estimated interaction energy [59].
Yet another approach to rational drug design involves probing the
osteocalcin crystal of the invention with molecules comprising a variety of
different functional groups to determine optimal sites for interaction between
candidate inhibitors and the protein. For example, high resolution x-ray
diffraction data collected from crystals soaked in or co-crystallized with
other
molecules allows the determination of where each type of solvent molecule
sticks. Molecules that bind tightly to those sites can then be further
modified
and synthesized and tested for their osteocalcin-hydroxyapatite
inhibitor/modulator activity [60].
In a related approach, iterative drug design is used to identify inhibitors of
osteocalcin. Iterative drug design i.s a method for optimizing associations
between a protein and a compound by determining and evaluating the
three-dimensional structures of successive sets of protein/compound
complexes. In iterative drug design, crystals of a series of protein/compound
complexes are obtained and then the three-dimensional structures of each
complex are solved. Such an approach provides insight into the association
between the proteins and compounds of each complex. This i s
accomplished by selecting compounds with inhibitory activity, obtaining
crystals of this new proteinlcornpound complex, solving the three
dimensional structure of the complex, and comparing the associations
between the new protein/compound complex and previously solved
proteinlcompound complexes. By observing how changes in the compound
34
CA 02446527 2003-10-23
affected the proteinlcompound associations, these associations may be
optimized.
A compound that is identified or designed as a result of any of these
methods can be obtained (or synthesized) and tested for its biological
activity, e.g., modulation of osteocalcin-hydroxyapatite binding.
Apparatus including the osteocalcin 3D structure or other osteocalcin
structural information
Storage media for the osteocalcin 3D structure or other osteocalcin
structural information include, but are not limited to: magnetic storage
media, such as floppy discs; hard disc storage medium, and magnetic tape;
optical storage media such as optical discs or CD-ROM; electrical storage
media such as RAM and ROM; and hybrids of these categories such as
magneticloptical storage media. Any suitable computer readable mediums
can be used to create a manufacture comprising a computer readable
medium having recorded on it an amino acid sequence and/or data of the
present invention.
"Recorded" refers to a process for storing information on computer readable
medium. A skilled artisan can readily adopt any of the presently know
methods for recording information on computer readable medium to store
an amino acid sequence, nucleotide sequence and/or EM data information
of the present invention.
A variety of data storage structures are available to a skilled artisan for
creating a computer readable medium having recorded thereon an amino
acid sequence andlor data of the present invention. The choice of the data
storage structure will generally be based on the means chosen to access
the stored information. In addition, a variety of data processor programs
and formats can be used to store the sequence and data information of the
present invention on computer readable medium. The sequence
information can be represented in a word processing text file, formatted in
CA 02446527 2003-10-23
commercially-available software such as WordPerfect and Microsoft Word,
or represented in the form of an ASCII file, stored in a database application,
such as DB2, Sybase, Oracle, or thf: like. A skilled artisan can readily adapt
any number of data processor structuring formats (e.g. text file or database)
in order to obtain computer readable medium having recorded thereon the
information of the present invention.
By providing the sequence and/or data on computer readable medium and
the structural information in this application, a skilled artisan can
routinely
access the sequence and data to model an osteocalcin, a subdomain
thereof, or a ligand thereof. As described above, computer algorithms are
publicly and commercially availablE, which allow a skilled artisan to access
this data provided in a computer readable medium and analyze it for
molecular modeling or other uses.
The present invention further provides systems, particularly computer-based
systems, which contain the sequence and/or data described herein. Such
systems are designed to do molecular modeling for an osteocalcin or at
least one subdomain or fragment thereof.
In one embodiment, the system includes a means for producing a 3D
structure of osteocalcin (or a fragment or derivative thereof) and means for
displaying the 3D structure of osteocalcin. The system is capable of
carrying out the methods described in this application. The system
preferably further includes a means for comparing the structural coordinates
of a candidate compound to the structural coordinates of the osteocalcin (or
a fragment or derivative thereof, such an active site or other region
described in this application) and means for determining if the candidate
compound is capable of modulating osteocalcin, as described in the
methods of the invention.
As used herein, "a computer-based system" refers to the hardware means,
software means, and data storage means used to analyze the sequence
and/or data of the present invention. The minimum hardware means of the
36
CA 02446527 2003-10-23
computer-based systems of the present invention comprises a central
processing unit (CPU), input means, output means, and data storage
means. A skilled artisan can readily appreciate which of the currently
available computer-based system are suitable for use in the present
invention.
As stated above, the computer-based systems of the present invention
comprise a data storage means having stored therein an osteocalcin or
fragment sequence andlor data of the present invention and the necessary
hardware means and software means for supporting and implementing an
analysis means. As used herein, "data storage means" refers to memory
which can store sequence or data (coordinates, distances, 3D structure
etc.) of the present invention, or a memory access means which can access
manufactures having recorded thereon the sequence or data of the present
invention.
As used herein, "search means" or "analysis means" refers to one or more
programs which are implemented on the computer-based system to
compare a target sequence or target structural motif with the sequence or
data stored within the data storage means. Search means are used to
identify fragments or regions of an osteocalcin which match a particular
target sequence or target motif. A variety of known algorithms are disclosed
publicly and a variety of commercially available software for conducting
search means are and can be used in the computer-based systems of the
present invention. A skilled artisan can readily recognize that any one of the
available algorithms or implementing software packages for conducting
computer analyses that can be adlapted for use in the present computer-
based systems.
As used herein, "a target structural motif," or "target motif," refers to any
rationally selected sequence or combination of sequences in which the
sequences(s) are chosen based on a three-dimensional configuration or
electron density map which is formed upon the folding of the target motif.
There are a variety of target motifs known in the art. Protein targets
include,
37
CA 02446527 2003-10-23
but are not limited to, active sites, structural subdomains, epitopes, and
functional domains. A variety of structural formats for the input and output
means can be used to input and output the information in the computer-
based systems of the present invention.
One application of this embodiment: provides a block diagram of a computer
system that can be used to implement the present invention. The computer
system includes a processor connected to a bus. Also connected to the
bus are a main memory (preferably implemented as random access
memory, RAM) and a variety of secondary storage memory such as a hard
drive and a removable storage medium. The removable medium storage
device may represent, for example, a floppy disk drive, A CD-ROM drive, a
magnetic tape drive, etc. A removalble storage unit (such as a floppy disk, a
compact disk, a magnetic tape, etc.) containing control logic and/or data
recorded therein may be inserted into the removable medium storage
medium. The computer system includes appropriate software for reading
the control logic andlor the data frorn the removable medium storage device
once inserted in the removable medium storage device. A monitor can be
used as connected to the bus to visualize the structure determination data.
Amino acid, encoding nucleotide or other sequence and/or data of the
present invention may be stored in a well known manner in the main
memory, any of the secondary storage devices, and/or a removable storage
device. Software for accessing and processing the amino acid sequence
and/or data (such as search tools, comparing tools, etc.) reside in main
memory during execution.
One or more computer modeling steps and/or computer algorithms are
used as described above to provide a molecular 3-D model, preferably
showing the 3D structure, of a cleaved osteocalcin, using amino acid
sequence data and atomic coordinates for the osteocalcin. The structure of
other osteocalcin-like molecules may be readily determined using methods
of the invention and the present knowledge of these molecules.
38
CA 02446527 2003-10-23
Accordingly, the invention provides computer media and systems for
performing a method of the invention. The invention includes a computer
readable media, such as a disk (eg. hard disk, floppy disk, CD-ROM, CD-
RW, DVD), with structural coordinate data of Table 3, recorded thereon,
osteocalcin bound to an inhibitor, substrate or a fragment of the foregoing
recorded thereon, the structure data being derivable from the structural
coordinate data of Table 3.The structural coordinate data is optionally
obtained by x-ray diffraction with a crystal of the invention.
Another aspect of the invention relates to a computer system, intended to
generate structures and/or perform rational drug design for osteocalcin, the
system containing structural coordinate data of Table 3, said data defining
the 3D structure of osteocalcin, ost~eocalcin bound to an inhibitor, substrate
or a fragment of the foregoing, said structure data being derivable from the
atomic coordinate data of Table 3. The structural coordinate data is
optionally obtained by x-ray diffractian with a crystal of the invention.
Osteocalcin Derivatives
The invention also includes the ud>e of osteocalcin derivatives to interfere
with osteocalcin binding to hydroxyapatite. Such derivatives include those
that retain the features on the hydroxyapatite binding surface but change the
features on the remaining surfaces such that osteocalcin will not interact
with cellslproteins in the original manner. Examples include:
(i) osteocalcin purified from non-human species, including but not limiting to
pig, monkey, cow, sheep, goat, dog, cat, rabbit, wallaby, rat, mouse,
xenopus, emu, chicken, carp, tetraodon, fugu, bluegill, seabream,
swordfish, other fish species, bird species, non-vertebrates
ii) mutating residues in 1-17 and/or 25-end
iii) insertion of residues into 1-17 and/or 25-end
iv) deletion of residues from 1-17 andlor 25-end
39
CA 02446527 2003-10-23
v) chemical modification of residues in 1-17 andlor 25-end using standing
chemical modification techniques.
In another embodiment, derivative are made such that only the features on
the hydroxyapatite binding surface are altered. This will direct the
osteocalcin derivatives to a location other than bone and allow it to compete
with the bone-bound osteocalcin in interacting with cellular protein and
reduce the recruitment of cell to bone. Such osteocalcin derivative may
include, but are not limited to
i) de-carboxylation of Glas by chemical means or by expressing osteocalcin
in host cells that cannot synthesize GLA.
ii) Mutation of residues on the surface, as can be deduced from the 3-D
structure (including but not limiting to Gla-17, Gla-21, Gla-24, Asp-30, Asp-
34), that can cause steric clash with the hydroxyapatite surface and therefore
prevent the OCN mutant from interacting with the HA surface.
In a further embodiment, bisphosphonate derivatives and tetracycline
derivatives may be used to bind the hydroxyapatite surface, thereby inhibiting
osteocalcin binding. Bisphosphonate and tetracycline derivatives have been
effective in treating osteoporosis, breast cancer and/or prostate cancer. The
main site of action of bisphosphonates and tetracycline derivative is likely
related to its ability to bind to hydroxyapatite. The exposed surface on
hydroxyapatite is likely mainly planar and therefore the interaction of
bisphosphonate and tetracycline ~nrill involve the planar surface on these
molecules. The planar surface has a lot of electronegative moieties that can
presumably interact with Ca and therefore bind to hydroxyapatite.
Compounds or derivatives of tetracycline and bisphosphonates can be
designed with an aim to:
(i) increase the planar surface area,
(ii) optimize its ineraction with hydroxyapatite surface
CA 02446527 2003-10-23
(iii) optimize its van der Waals interaction with the hydroxyapatite surface
and
(iv) optimize its electrostatic interaction with the hydroxyapatite surface.
Examples of bisphosphonates that can be used or modified, include but are
not limited to, pyrophosphate, bisphosphonate, etidronate, clodronate,
pamidronate, tiludronate, risedronate, zoledronate, alendronate, YM-175,
and ibandronate. The two phosphates on all bisphosphonates are located
on one side of the molecule. The design of a new compound would
therefore involve addition of phosphates or other electronegative moieties to
the carbon linking the two phosphates or to any other atoms on the structure
such that the newly added phosphate or electronegative moiety will be
positioned on the same plane as the two existing phosphates.
The design of compounds may achieve at least one of the following
properties:
(i) bind to hydroxyapatite tighter than osteocalcin
(ii) have less toxicity
(iii) displace osteocalcin more efficiently.
Assays of osteocalcin or other derivatives or inhibitors identified from the
osteocaicin structure
Once identified, the inhibitor may then be tested for bioactivity using
standard techniques (e.g. in vitro or in vivo assays). For example, the
compound identified by drug design may be used in binding assays using
conventional formats to screen agonists (e.g by measuring in vivo or in vitro
binding of osteocalcin after addition of a compound). Suitable assays
include, but are not limited to, the enzyme-linked immunosorbent assay
(ELISA), or a fluorescence quench assay. In evaluating osteocalcin
modulators for biological activity in animal models (e.g. rat, mouse, rabbit),
41
CA 02446527 2003-10-23
various oral and parenteral routes of administration are evaluated.
The method may also comprise obtaining or synthesizing the compound
and determining whether the compound modulates the activity of the
osteocalcin, fragment or derivative in an in vivo or in vitro assay. Such an
assay optionally comprises:
a) obtaining osteocalcin with recombinant technology, chemical synthesis
or purification from bone;
b) contacting osteocalcin with hydroxyapatite;
c) adding a test compound to compete with the bound osteocalcin for
hydroxyapatite; and
d) measuring the amount of osteocalcin dissociated from hydroxyapatite as
a result of the addition of the test compound;
whereby a compound that competes with osteocalcin for hydroxyapatite is
identified as a compound that inhibits osteocalcin activity.
Preferably, inhibitors may be used in a screening assay involving the
following steps:
(i) Label osteocalcin with fluorescence
(ii) incubate labeled osteocalcin with hydroxyapatite powder
(iii) wash off excess osteocalcin
(iv) add different concentrations of the compound to be analysed
(v) measure the release of osteocalcin from the hydroxyapatite-osteocalcin
complex by an appropriate spectrophotometer, for example, fluorexcence
spectrophotometer, fluorescence polarization spectrophotometer) and
(vi) determine the potency of the compound in releasing osteocalcin.
42
CA 02446527 2003-10-23
Furthermore, this screening assay may be carried out in a High Throughput
manner using a robotic system.
Pharmaceuticalldiagnostic formulations, methods of medical treatment
and uses
Medical Treatments and Uses
Abnormal bone turnover causes many diseases in mammals, such as
humans. Examples of these diseases include: bone metabolic disorders,
osteoporosis, breast cancer, prostate cancer, lung cancer and hypercalcemia
malignancy.
Accordingly, the invention includes a method of medical treatment of a
disease, preferably bone disease, in a subject having bone metabolic
disorders, osteoporosis, breast cancer, prostate cancer, lung cancer and
hypercalcemia, comprising administering to the subject a compound or
derivative of the invention or a compound identified by a method of the
invention. The invention also includes the use of such
compoundslderivatives for treatment of a disease, preferably bone disease,
in a subject having bone metabolic disorders, osteoporosis, breast cancer,
prostate cancer, lung cancer and hypercalcemia.
Pharmaceutical Compositions
Inhibitors may be combined in pharmaceutical compositions according to
known techniques. The compounds/derivatives are preferably incorporated
into pharmaceutical dosage forms suitable for the desired administration
route such as tablets, dragees, capsules, granules, suppositories,
solutions, suspensions and lyophilized compositions to be diluted to obtain
injectable liquids. The dosage forms are prepared by conventional
techniques and in addition to the inhibitor could contain solid or liquid
inert
diluents and carriers and pharmaceutically useful additives such as lipid
vesicles liposomes, aggregants, disaggregants, salts for regulating the
osmotic pressure, buffers, sweeteners and colouring agents. Slow release
43
CA 02446527 2003-10-23
pharmaceutical forms for oral use may be prepared according to
conventional techniques. Other pharmaceutical formulations are described
for example in US 5,192,746.
Pharmaceutical compositions used to treat patients having diseases,
disorders or abnormal physical states could include a compound of the
invention and an acceptable vehicle or excipient [61] and subsequent
editions). Vehicles include saline and D5W (5~/o dextrose and water).
Excipients include additives such as a buffer, solubifizer, suspending agent,
emulsifying agent, viscosity controlling agent, flavor, lactose filler,
antioxidant, preservative or dye. The compound may be formulated in solid
or semisolid form, for example pills, tablets, creams, ointments, powders,
emulsions, gelatin capsules, capsules, suppositories, gels or membranes.
Routes of administration include oral, topical, rectal, parenteral
(injectable),
local, inhalant and epidural administration. The compositions of the
invention may also be conjugated to transport molecules to facilitate
transport of the molecules. The methods 'for the preparation of
pharmaceutically acceptable compositions which can be administered to
patients are known in the art.
The pharmaceutical compositions can be administered to humans or
animals. Dosages to be administered depend on individual patient
condition, indication of the drug, physical and chemical stability of the
drug,
toxicity, the desired effect and on the chosen route of administration [62].
The present invention has been described in detail and with particular
reference to the preferred embodiments; however, it will be understood by
one having ordinary skill in the art that changes can be made thereto without
departing from the spirit and scope thereof.
Methods
Protein Production and Purification
44
CA 02446527 2003-10-23
Osteocalcin was extracted from its natural source, bone, and purification
was carried out based on the previously described protocol (21 ] with
modification for scale-up production. The diaphysis of femur bone was
separated from the epiphysis with a band saw and flesh was removed from
the diaphysis with a razor blade and wood scraper. After soaking in cold
acetone for 10 minutes, the periosteum lining was removed with a wood
scraper and steel wool. The marrow was subsequently removed with a long
spatula and the medullary cavity was cleaned with a test tube brush in warm
soap water. The cleaned bone diaphysis was cut longitudinally in half then
crosscut into thin slices (~2 mm) then frozen in liquid nitrogen and
lyophilized overnight (FTS Systems Inc.). The lyophilized bone was frozen in
liquid nitrogen then ground into powder with a stainless steel blender
(blaring Commercial Blender) followed by a coffee grinder (Braun). The
bone powder was sieved through a stainless steel mesh yielding bone
powder with average size of 200 micro meter. The fine powder (30 grams)
was washed two times with 500 ml of cold water containing 0.6 mM PMSF at
4°C for 30 minutes and the pellet was collected after centrifugation
(Sorvall)
at 1500 x g for 30 minutes. The pellet was frozen in liquid nitrogen and
lyophilized. The freeze-dried powder was demineralized at 4°C by gentle
stirring for 4 hours in 300 ml of 20% formic acid {HCOOH) containing 0.6
mM PMSF. The solution was then centrifuged at 40,000 x g for 60 minutes.
The insoluble pellet was resuspended with 50 mt of 20% formic acid, stirred
for 30 minutes to release any trapped proteins, and recentrifuged. The
40,000 x g supernatants were combined and filtered (Millipore AP2004700).
The filtered supernatant was made to 0.1 % Trifluoroacetic Acid (CF3COOH)
(TFA). Sep-Pak C18 cartridges (Waters No. WAT043345) were mounted
onto a 1-liter flask under vacuum, conditioned with sequential addition of
100 ml Methanol (CH3OH) and 100 ml of 0.1 % TFA, and then loaded with
100 ml aliquots of the filtered supernatant. After sequential washings with
100 ml of 0.1 % TFA and 100 ml of 30% methanol in 0.1 % TFA, the bound
material was eluted with 25 ml of 80% methanol in 0.1 % TFA into a flask
containing 10 micro liter of 300 mM PMSF. The flow rate was set at 5
CA 02446527 2003-10-23
mllminute in the load and elution steps and 10 ml/minute in other steps.
The eluate was concentrated to about 20 ml with a speed vac (Savant Sped-
Vac SC210A). The concentrated solution was further purified with reverse
phase high performance liquid chromatography (HPLC). The HPLC system
consisted of Waters Delta Pak C18-100A 19mm x 30cm column mounted
onto Beckman pumps (Beckman 112 Solvent Delivery Module), which are
controlled by a BioRad Chromotograph software on Windows 95. The
elution gradient was created by mixture of two eluants, eluant A consisted of
HPLC grade acetonitrile (ACN) and eluant B consisted of 0.1 % TFA. The
gradient profile was set from 25% to 45% ACN over 30 minutes. The flow
rate was set at 10 ml/minute. The well-resolved peak that eluted after 40
minutes (39% ACN) was collected, frozen in liquid nitrogen and lyophilized
overnight. The freeze-dried protein was stored at -2U °C.
Crystallization
Crystallization was performed by the vapor diffusion method [22) in hanging
drop mode for crystal screening and in sitting drop mode for diffraction
quality crystals. For hanging drops, a small bead of grease was placed on
the rim of each well in the crystallization tray. Typically 500 ml of mother
liquor was placed into the reservoir. The purified protein was dissolved in
solution to 10 mg/ml and 2 micro liter of the protein solution was mixed with
2 micro liter of mother liquor on the silanized cover slip. The cover slip was
then sealed over the well in the inverted position, such that the mother
liquor
and the drop on the cover slip share the same air space.
The crystals used in the structure determination were grown with a reservoir
containing 0.1M HEPES pH 7.5, 10 mM CaCl2 and 10% wlv PEG 4000.
Crystals appeared within two weeks at room temperature and reached a
maximum size of 0.2 times 0.2 times 0.6 mm.
Data Collection
46
CA 02446527 2003-10-23
Single anomalous scattering (SAS) data were collected at beamline IMCA
CAT ID-17 of the Advanced Photon Source at Argonne National Laboratory.
The x-ray wavelength was set at 1.7 A to maximize the anomalous signals.
Osteocalcin crystals were flash frozen in a nitrogen cold stream after
transferred to a cryo-protectant solution containing 30% PEG 4000, 10 mM
CaCl2, and 0.1 M HEPES at pH 7.4. The x-ray detector, Mar Research m 165
CCD, was placed 50 mm from the crystal. Data was collected in oscillation
mode from 0 to 180 degree and diffraction images were recorded for each
degree of rotation at 3.0 sec per frame. The crystal was kept frozen at -160
°C during data collection using the Oxford cryosystem cooling device.
The
intensities of the diffraction data were integrated and indexed using DENZO
and reduced using SCALEPACK of the HKL package [23]. The spacegroup
was P3121, with cell dimensions a=b=54 Angstrom and c=35 Angstrom.
Structure Determination and Refinement
The positions of calaum and sulfur atoms were located using Bij~oet
difference Pat6erson functions.Automated Patterson search as well as phase
determination were performed with the program SOLiiE [24].
Solent flattening was carried outwith the program RESOLVE (25].
The program O [26] was used for model building and rebuilding. Model
building was initiated using the 2.0 ~, electron density map calculated with
SAS phases thatwere improved by solvent flattening. Refinement was carried
out with the program CNS (version 1.11) [27]. Positional and simulated
annealing refinement with ma~amum likelihood targets was carried out after
rigid body refinement. Iterative cycles of model building and refinementwere
carried out until the x ray residual facfi~r, R-factor, was stationary and no
more
information can be obtained from SigmaA weighted [28] 2~Fos - ~Fc) electron-
density map, difference map (Fo- Fc), and omitmap. The model consists of
all residues for the porane osteocalcin, except a missing N-term us region
(residues 1 to 12). The R-factor is 25% and the free R is 28% for reflections
in
the interval 30-2.0 Angstrom. 93% of the residues fall in the most favorable
47
CA 02446527 2003-10-23
regions of the Ramachandran plot as defined by Procheck. Data collection
and refinement statistics are summarized in Table 2. The crystal structure
giving the atomic structural coordinates is given in Table 3.
48
CA 02446527 2003-10-23
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52
CA 02446527 2003-10-23
Table 1. Amino acid sequence of osteocalcin.
Monkey YLYQW LGAPA PYPDP LEPKR EVCEL NPDCD ELADH IGFQE AYRRF YGPV
Rabbit QLING QGAPA PYPDP LEPKR EVCEL NPDCD ELADQ VGLQD AYQRF YGPV
Human YLYQW LGAPA VYPDP LEPRR EVCEL NPDCD ELADH IGFQE AYRRF YGPV
1
Cow YLDHW LGAPAPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPV
Pig YLDHG LGAPAPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGIA
Sheep YLDPG LGAPAPYPDPLEPRREVCELNPDCDELADHIGFQEAYRRFYGPV
Goat YLDPG LGAPAPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPV
Dog YLDSG LGAPVPYPDPLEPKREVCELNPNCDELADHIGFQEAYQRFYGPV
Cat YLAPG LGFPAPYPDPLEPKREICELNPDCDELADHIGFQDAYRRFYGTV
WallabyYLYQT LGAPFPYPDPQENKREVCELNPDCDELADHIGFSEAYRRFYGTA
Rat YLNNG LGAPAPYPDPLEPHREVCELNPNCDELADHIGFQDAYKRIYGTTV
Mouse YL GASV PSPDPLEPTREQCELNPACDELSDQYGLKTAYKRIYGITI
XenopusSYGNN VGQGAAVGSPLESQREVCELNPDCDELADHIGFQEAYRRFYGPV
Emu SFAV GSSYGAAPDPLEAQREVCELNPDCDELADHIGFQEAYRRFYGPV
ChickenHYAQDSGVAGAPYPDPLEPKREVCELNPDCDELADHIGFQEAYRRFYGPV
Carp AG TAPADLTVAQLESLKEVCEANLACEHMMDVSGIIAAYTAYGPIPY
Tetraodon AAGE PTLQQLESLREVCELNIACDEMADPAGIVAAYAAYYGPPT
F
Fugu APGE PTPQQLESLREVCELNIACDEMADTAGIVAAYAAYYGPPP
F
Bluegill RAGE LTLTQLESLREVCEANLACEDMMDAQGIIAAYTAYYGPIP
Y
Seabream AAGQ LSLTQLESLREVCELNLACEHMMDTEGIIAAYTAYYGPIP
Y
Swordfish TRAGDLTPLQLESLREVCELNVSCDEMADTAGIVAAYIAYYGPIQ
A F
CA 02446527 2003-10-23
Table 2. Statistics of data reduction and refinement.
Data collection
2
Space group 1'3,21
Data set Crystal 1 Crystal 2 Combined
Wavelength (t~) 1.70 71.70
Resolution (.~) 27.7-2.0 27.7-2.0
(2.07-2.00)' (2.07-2.00)
1
Completeness (%) 97.0 (94.3)' ~>3.1 (92.2)'95.9 (93.8)'
Redundancy 17 (>_ 4)' 1_2 (>_ 2)' 23 (>_
5)'
Rmer~e2 4.9 (21.4)' 4.8 (13.3)' 6.7 (21.9)'
I/a(I) 14.7 (5.35)' 25.8 (9.90)'25.1 (16.5)'
Refinement
Unique reflection 6230
Protein atoms 314
Solvent atoms 60
R-work (Rfree) (%)3 25.5 (28.3)
Average B-factor (t~rz)37.1
Deviation from ideal
geometry
Bonds (A) 0.006
Angles () 1.3
Ramachandran plot
Most favored regions (%) 93.3
Additionally allowed regions (%) 2
Generously allowed regions (%) 0
Disallowed regions (%) 0
'Highest resolution shell.
2 Rmerge = EnE~ ~In~ <Ih>~~<In>, where Ih~ is the intensity of the i~'
observation of reflection h, and <Ih>~ is
the average intensity of redundant measurements of the h reflections.
3R-work = E~~Fo~-~F~II~~IFoI~ where Fo and F~ are the observed and calculated
structure
factor amplitudes. Rfree is monitored with 948 reflections excluded from
refinement.
CA 02446527 2003-10-23
Table 3. Coordinate of porcine osteocalcin
REMARK 3
REMARK 3 REFINEMENT.
REMARK 3 PROGRAM . CNS l.i
REMARK 3 AUTHORS . BRUNGER, ADAMS, CLORE, DELANO,
REMARK 3 EROS, GROSSE-KUNSTLEVE, J:CANG,
REMARK 3 KUSZEWSKI, NILGES, PANNU, READ,
REMARK 3 RICE, SIMONSON, WARREN
REMARK 3
REMARK 3 DATA USED IN REFINEMENT.
REMARK 3 RESOLUTION RANGE HIGH (ANGSTROMS) . 2.00
REMARK 3 RESOLUTION RANGE LOW (ANGSTROMS) . 27.72
REMARK 3 DATA CUTOFF (SIGMA(F)) . 3.0
REMARK 3 DATA CUTOFF HIGH (ABS(F)) . 462281.14
REMARK 3 DATA CUTOFF LOW (ABS(F)) . 0.000000
REMARK 3 COMPLETENESS (WORKING+TEST) (%) . 87.'7
REMARK 3 NUMBER OF REFLECTIONS . 6230
REMARK 3
REMARK 3 FIT TO DATA USED IN REFINEMENT.
REMARK 3 CROSS-VALIDATION METHOD . THROUGHOUT
REMARK 3 FREE R VALUE TEST SET SELECTION . RANDOM
REMARK 3 R VALUE (WORKING SET) . 0.255
REMARK 3 FREE R VALUE . 0.283
REMARK 3 FREE R VALUE TEST SET SIZE (%) . 15.2
REMARK 3 FREE R VALUE TEST SET COUNT . 94E3
REMARK 3 ESTIMATED ERROR OF FREE R VALUE . 0.009
REMARK 3
REMARK 3 FIT IN THE HIGHEST RESOLUTION BIN.
REMARK 3 TOTAL NUMBER OE' BINS USED . 6
REMARK 3 BIN RESOLUTION RANGE HIGH (A) . 200
REMARK 3 BIN RESOLUTION RANGE LOW (A) . 2..13
REMARK 3 BIN COMPLETENESS (WORKING+TEST) (%) . 6E3.6
REMARK 3 REFLECTIONS IN BIN (WORKING SET) . 704
REMARK 3 BIN R VALUE (WORKING SET) . 0.306
REMARK 3 BIN FREE R VALUE . 0.400
REMARK 3 BIN FREE R VALUE TEST SET SIZE
(%) 12.9
REMARK 3 BIN FREE R VALUE TEST SET COUNT . 104
REMARK 3 ESTIMATED ERROR OF BIN FREE R VALUE : 0.039
REMARK 3
REMARK 3 NUMBER OF NON-HYDROGEN ATOMS USED IN REF7:NEMENT.
REMARK 3 PROTEIN ATOMS . 0
REMARK 3 NUCLEIC ACID ATOMS . 0
REMARK 3 HETEROGEN ATOMS . 0
REMARK 3 SOLVENT ATOMS . 0
REMARK 3
REMARK 3 B VALUES.
REMARK 3 FROM WILSON PLOT (A**2) . l7.Ei
REMARK 3 MEAN B VALUE (OVERALL, A**2) . 37.1.
REMARK 3 OVERALL ANISOTROPIC B VALUE.
REMARK 3 B11 (A**2) . 2.05
REMARK 3 B22 (A**2) . 2.05
REMARK 3 B33 (A**2) . -4.10
REMARK 3 B12 (A**2) . 3.91
REMARK 3 B13 (A**2) . 0.00
REMARK 3 B23 (A**2) . 0.00
REMARK 3
REMARK 3 BULK SOLVENT MODELING.
REMARK 3 METHOD USED : E'LAT MODEL
REMARK 3 KSOL . 0.349568
REMARK 3 BSOL . 32.3123 (A**2)
REMARK 3
REMARK 3 ESTIMATED COORDINATE ERROR.
REMARK 3 ESD FROM LUZZATI PLOT
(A) . 0.31
REMARK 3 ESD FROM SIGMAA. (A) . 0.27
REMARK 3 LOW RESOLUTION CUTOFF (A) . 5.00
3
CA 02446527 2003-10-23
REMARK3
REMARK3 CROSS-VALIDATEDTIMATED COORDINATE
ES ERROR.
REMARK3 ESD FROM C-V ATI PLOT (A) . 0.36
LUZZ
REMARK3 ESD FROM C-V
SIGMAA (A)
. 0.35
REMARK3
REMARK3 RMS DEVIATIONS
FROM IDEAL
VALUES.
REMARK3 BOND LENGTHS (A) . 0.006
REMARK3 BOND ANGLES (DEGREES) . 1.3
REMARK3 DIHEDRAL ANGLES(DEGREES) . 20.5
REMARK3 IMPROPER ANGLES(DEGREES) . 0.99
REMARK3
REMARK3 ISOTROPIC THERMALMODEL : RESTRAINED
REMARK3
REMARK3 ISOTROPIC THERMALFACTOR RESTRAINTS. S SIGMA
RM
REMARK3 MAIN-CHAIN (A**2) . NULL , NULL
BOND
REMARK3 MAIN-CHAIN (A**2) . NLJLL , NULL
ANGLE
REMARK3 SIDE-CHAIN (A**2) . NULL , NULL
BOND
REMARK3 SIDE-CHAIN (A**2) . NIJLL , NULL
ANGLE
REMARK3
REMARK3 NCS MODEL :
NONE
REMARK3
REMARK3 NCS RESTRAINTS.I2MS SIGMA/WEIGHT
REMARK3 GROUP 1 POSITIONAL (A) . NULL , NULL
REMARK3 GROUP 1 B-FACTOR (A**2) . N(JLL , NULL
REMARK3
REMARK3 PARAMETER FILE. CNS_TOPPAR/protein_rep.param
1
REMARK3 PARAMETER FILE. CNS_TOPPAR/dna-rna_r_ep.param
2
REMARK3 PARAMETER FILE. CNS_TOPPAR/water_rep.param
3
REMARK3 PARAMETER FILE. CNS_TOPPAR/ion.pararn
4
REMARK3 TOPOLOGY FILE . CNS_TOPPAR/protein.top
_
REMARK3 TOPOLOGY FILE . CNS_TOPPAR/dna-rna.i=op
2.
REMARK3 TOPOLOGY FILE . CNS_TOPPAR/water.top
3
REMARK3 TOPOLOGY FILE . CNS_TOPPAR/ion.top
4
REMARK3
REMARK3 OTHER REFINEMENT
REMARKS: NULL
SEQRES1 A 101 PRO ASP LEU CGU PRO ARG VAL CYS LEU
PRO ARG CGU CGU
SEQRES2 A 101 ASN PRO CYS ASP GLU LEU HIS ILE PHE
ASP ALA ASP GLY
SEQRES3 A 101 GLN GLU TYR ARG ARG PHE ILE ALA CA2
J1LA TYR GI~Y CA2
SEQRES4 A 101 CA2 TIP TIP TIP TIP TIP TIP TIP TIP
7.'IP TIP T==P TIP
SEQRES5 A 101 TIP TIP TIP TIP TIP TIP TIP TIP TIP
TIP TIP T:CP TIP
SEQRES6 A 101 TIP TIP TIP TIP TIP TIP TIP TIP TIP
TIP TIP TIP TIP
SEQRES7 A 101 TIP TIP TIP TIP TIP TIP TIP TIP TIP
TIP TIP TIP TIP
SEQRES8 A 101 TIP TIP TIP TIP TIP TIP TIP
TIP TIP TIP
SSBOND1 CYS A 23 CY:i 29
A
CRYST151 .491 51.491 389 90.00 90.00 P 31 2 6
35. 120.00 1
ORIGXl 1.000000 0.0000000.000000 0.00000
ORIGX2 0.000000 1.0000000.000000 0.00000
ORIGX3 0.000000 0.0000001.000000 0.00000
SCALE1 0.019421 0.0112130.000000 0.00000
SCALE2 0.000000 0.0224250.000000 0.00000
SCALES 0.000000 O.OOOG000.028257 0.00000
ATOM 1 CB PRO A 13 8.383 28.488 44.4341.00 37.68
ATOM 2 CG PRO A 13 7.919 29.624 45.3361.00 36.60
ATOM 3 C PRO A 13 9.566 29.662 42.5411.00 37.52
ATOM 4 0 PRO A 13 9.275 30.855 42.4441.00 38.00
ATOM 5 N PRO A 13 10.210 29.966 44.9351.00 38.06
ATOM 6 CD PRO A 13 9.196 30.126 45.9951.00 36.47
ATOM 7 CA PRO A 13 9.718 29.013 43.97.91.00 37.33
ATOM 8 N ASP A 14 9.777 28.879 41.4831.00 36.83
ATOM 9 CA ASP A 14 9.671 29.384 40.17_61.00 36.7.3
ATOM 10 CB ASP A 14 10,607 28.596 39.2041.00 40.35
ATOM 11 CG ASP A 14 10.728 29.211 37.8241.00 43.98
ATOM 12 OD1 ASP A 14 9.681 29.481 37.1921.00 44.48
ATOM 13 OD2 ASP A 14 11.874 29.430 37.3711.00 47.64
ATOM 14 C ASP A 14 8.232 29.268 39.6011.00 33.88
ATOM 15 O ASP A 14 7.721 28.169 39.47.31.00 33.66
ATOM 16 N PRO A 15 7.570 30.409 39.3491.00 30.77
ATOM 17 CD PRO A 15 8.106 31.776 39.4681.00 31.26
4
CA 02446527 2003-10-23
ATOM 18 CA PROA 15 6.189 30.43338.8561.0029.44
ATOM 19 CB PROA 15 5.865 31.92838.8191.0029.57
ATOM 20 CG PROA 15 7.181 32.56238.5711.0028.96
ATOM 21 C PROA 15 5.990 29.75637.4971.0027.79
ATOM 22 0 PROA 15 4.870 29.39637.1321.0023.99
ATOM 23 N LEUA 16 7.088 29.57036.7631.0027.93
ATOM 24 CA LEUA 16 7.053 28.95135.4451.0026.79
ATOM 25 CB LEUA 16 8.196 29.50234.5861.0028.95
ATOM 26 CG LEUA 16 8.067 30.96134.1371.0028.97
ATOM 27 CD1LEUA 16 9.374 31.41033.5191.0031.78
ATOM 28 CD2LEUA 16 6.934 31.11133.1441.0028.68
ATOM 29 C LEUA 16 7.118 27.42435.4451.0025.59
ATOM 30 O LEUA 16 6.950 26.80934.3981.0023.04
ATOM 31 N CGUA 17 7.359 26.81836.6061.0024.70
ATOM 32 CA CGUA 17 7.453 25.36036.7021.0025.21
ATOM 33 CB CGUA 17 7.547 24.92438.1631.0028.34
ATOM 34 C CGUA 17 6.252 24.66636.0601.0024.08
ATOM 35 O CGUA 17 6.408 23.69835.3271.0022.85
ATOM 36 CG CGUA 17 8.807 24.09038.5251.0029.46
ATOM 37 CD1CGUA 17 9.396 23.28637.3361.0028.04
ATOM 38 CD2CGUA 17 8.411 23.25539.7401.0032.29
ATOM 39 OE1CGUA 17 10.339 23.77536.6901.0031.46
ATOM 40 OE2CGUA 17 8.917 22.16037.0751.0026.97
ATOM 41 OE3CGUA 17 7.958 23.92640.6681.0035.00
ATOM 42 OE4CGUA 17 8.527 22.03639.7801.0033.69
ATOM 43 N PROA 18 5.029 25.13536.3491.0023.16
ATOM 44 CD PROA 18 4.584 26.06437.4041.0023.03
ATOM 45 CA PROA 18 3.884 24.47035.7271.0023.07
ATOM 46 CB PROA 18 2.705 25.29536.2331.0023.16
ATOM 47 CG PROA 18 3.143 25.65437.6061.0021.79
ATOM 48 C PROA 18 3.985 24.42934.1961.0022.93
ATOM 49 0 PROA 18 3.746 23.38833.5901.0023.19
ATOM 50 N ARGA 19 4.339 25.55133.5731.0018.14
ATOM 51 CA ARGA 19 4.467 25.57532.1231.0019.74
ATOM 52 CB ARGA 19 4.611 27.01131.6141.0019.63
ATOM 53 CG ARGA 19 3.310 27.81431.6961.0023.45
ATOM 54 CD ARGA 19 3.424 29.11830.9481.0025.21
ATOM 55 NE ARGA 19 2.132 29.78430.8221. 29.06
U0
ATOM 56 CZ ARGA 19 1.921 30.85730.0651.0028.89
ATOM 57 NH1ARGA 19 2.919 31.38529.3681.0028.53
ATOM 58 NH2ARGA 19 0.712 31.39229.9991.0029.82
ATOM 59 C ARGA 19 5.647 24.72931.6:381.0019.99
ATOM 60 O ARGA 19 5.571 24.09130.5831.0019.51
ATOM 61 N ARGA 20 6.737 24.73132.4041.0022.01
ATOM 62 CA ARGA 20 7.902 23.94332.0521.0022.37
ATOM 63 CB ARGA 20 9.024 24.13633.0671.0026.75
ATOM 64 CG ARGA 20 9.586 25.54133.1151.0032.44
ATOM 65 CD ARGA 20 10.812 25.59734.0()01.0036.42
ATOM 66 NE ARGA 20 11.528 26.85333.8111. 43.51
CO
ATOM 67 CZ ARGA 20 12.749 27.09834.2'791.0047.68
ATOM 68 NH1ARGA 20 13.402 26.16934.9'711.0049.20
ATOM 69 NH2ARGA 20 13.323 28.27134.0451.0047.89
ATOM 70 C ARGA 20 7.515 22.46832.O:L91.0024.90
ATOM 71 0 ARGA 20 7.956 21.73831.1301.0024.00
ATOM 72 N CGUA 21 6.701 22.02232.9801.0024.22
ATOM 73 CA CGUA 21 6.293 20.61233.0121.0023.24
ATOM 74 CB CGUA 21 5.506 20.26734.2f391.0024.58
ATOM 75 C CGUA 21 5.432 20.29331.8051.0023.70
ATOM 76 0 CGUA 21 5.561 19.22131.2161.0020.30
ATOM 77 CG CGUA 21 6.392 20.44535.5:?81.0026.52
ATOM 78 CDlCGUA 21 7.353 19.24935.7541.0027.96
ATOM 79 CD2CGUA 21 5.507 20.71836.7381.0029.78
ATOM 80 OE1CGUA 21 8.366 19.40636.4821.0027.23
ATOM 81 OE2CGUA 21 7.056 18.15935.2171.0025.25
ATOM 82 OE3CGUA 21 4.695 21.62536.5861.0036.91
ATOM 83 OE4CGUA 21 5.664 20.13937.7x71.0032.02
ATOM 84 N VALA 22 4.553 21.22631.4411.0020.53
ATOM 85 CA VALA 22 3.678 21.03130.2921.0021.98
CA 02446527 2003-10-23
ATOM86 CB VALA 22 2.762 22.26830.0621.0023.28
ATOM87 CGlVALA 22 2.078 22.18028.7071.0024.34
ATOM88 CG2VALA 22 1.726 22.36331.1661.0020.77
ATOM89 C VALA 22 4.536 20.79529.0501.0022.61
ATOM90 0 VALA 22 4.319 19.83228.3051.0023.17
ATOM91 N CYSA 23 5.523 21.66128.8461.0022.34
ATOM92 CA CYSA 23 6.423 21.56627.6991.0024.86
ATOM93 C CYSA 23 7.212 2D.24827.6921.0025.63
ATOM94 O CYSA 23 7.306 19.59926.6561.0022.02
ATOM95 CB CYSA 23 7.380 22.77127.6891.0025.85
ATOM96 SG CYSA 23 8.527 22.92126.2621.0030.27
ATOM97 N CGUA 24 7.761 19.85228.8421.0026.69
ATOM98 CA CGUA 24 8.527 18.60728.9311.0029.70
ATOM99 CB CGUA 24 8.981 18.30430.3'071.0026.05
ATOM100 C CGUA 24 7.665 17.45628.4761.0031.08
ATOM101 0 CGUA 29 8.143 16.54127.8121.0032.94
ATOM102 CG CGUA 24 9.966 19.35730.8'761.0026.18
ATOM103 CD1CGUA 24 11.275 19.29030.0931. 24.75
U0
ATOM104 CD2CGUA 24 10.148 19.17232.3901.0027.43
ATOM105 OE1CGUA 24 12.023 18.29330.2331.0029.79
ATOM106 OE2CGUA 24 11.537 20.24429.3481.0024.99
ATOM107 OE3CGUA 24 9.100 19.19033.0431.0028.87
ATOM108 OE4CGUA 24 11.260 19.08432.9081.0024.87
ATOM109 N LEUA 25 6.392 17.50728.8501.0032.75
ATOM110 CA LEUA 25 5.445 16.45828.5061.0036.12
ATOM111 CB LEUA 25 4.089 16.76129.1371.0036.84
ATOM112 CG LEUA 25 3.183 15.54929.3521.0036.31
ATOM113 CD1LEUA 25 3.839 14.61430.3621.0036.20
ATOM114 CD2LEUA 25 1.821 15.99929.8541.0034.69
ATOM115 C LEUA 25 5.292 16.30826.9931.0036.64
ATOM116 0 LEUA 25 5.064 15.20726.4901.0038.56
ATOM117 N ASNA 26 5.411 17.42226.2781.0037.58
ATOM118 CA ASNA 26 5.304 17.43024.8211.0038.83
ATOM119 CB ASNA 26 4.709 18.75924.3401.0040.62
ATOM120 CG ASNA 26 4.386 18.75722.8491.0042.54
ATOM121 OD1ASNA 26 5.213 18.38122.0141.0041.62
ATOM122 ND2ASNA 26 3.179 19.19322.5111.0042.90
ATOM123 C ASNA 26 6.697 17.24624.2101.0038.32
ATOM124 O ASNA 26 7.494 18.18524.1711.0036.32
ATOM125 N PROA 27 7.004 16.03423.7161.0039.18
ATOM126 CD PROA 27 6.127 14.86823.5281.0039.15
ATOM127 CA PROA 27 8.318 15.79123.1191.0039.23
ATOM128 CB PROA 27 8.135 14.45222.4011.0039.42
ATOM129 CG PROA 27 6.646 14.32222.2401.0040.53
ATOM130 C PROA 27 8.759 16.90722.1881.0040.17
ATOM131 0 PROA 27 9.897 17.38622.2641.0040.66
ATOM132 N ASPA 28 7.847 .17.34121.3281.0039.89
ATOM133 CA ASPA 28 8.149 18.40120.3831.0038.18
ATOM134 CB ASPA 28 6.943 18.63819.4721.0041.42
ATOM135 CG ASPA 28 6.535 17.38618.7211.0041.42
ATOM136 OD1ASPA 28 7.426 16.74718.1301.0042.81
ATOM137 OD2ASPA 28 5.333 17.04218.7201.0043.97
ATOM138 C ASPA 28 8.552 19.69721.0741.0037.41
ATOM139 O ASPA 28 9.459 20.39920.6111.0035.62
ATOM140 N CYSA 29 7.875 20.02222.1741.0034.99
ATOM141 CA CYSA 29 8.203 21.23622.91.01.0034.20
ATOM142 C CYSA 29 9.487 20.97523.6911.0032.10
ATOM143 0 CYSA 29 10.353 21.84123.7891.0030.74
ATOM144 CB CYSA 29 7.080 21.63023.8911.0032.50
ATOM145 SG CYSA 29 7.340 23.27324.69:41.0032.82
ATOM146 N ASPA 30 9.604 19.77024.2341.0032.83
ATOM147 CA ASPA 30 10.776 19,40225.01.41.0034.15
ATOM148 CB ASPA 30 10.685 17.94925.4641.0033.18
ATOM149 CG ASPA 30 11.714 17.60726.52.31.0032.22
ATOM150 OD1ASPA 30 12.621 18.42826.7521.0032.53
ATOM151 OD2ASPA 30 11.608 16.52427.1251.0031.78
ATOM152 C ASPA 30 12.026 19.58024.1771.0036.29
ATOM153 O ASPA 30 12.937 20.32224.5441.0034.50
6
CA 02446527 2003-10-23
ATOM154 N GLUA 31 12.056 18.88523.0451.0039.34
ATOM155 CA GLUA 31 13.186 18.95422.1351.0040.16
ATOM156 CB GLUA 31 12.901 18.12420.8831.0042.69
ATOM157 CG GLUA 31 13.972 18.25119.8131.0045.38
ATOM158 CD GLUA 31 15.358 17.95220.3451.0045.54
ATOM159 OE1GLUA 31 15.566 16.82520.8471.0044.75
ATOM160 OE2GLUA 31 16.230 18.84520.2601.0044.69
ATOM161 C GLUA 31 13.483 20.39421.7441.0040.61
ATOM162 0 GLUA 31 14.609 20.86321.8861.0041.73
ATOM163 N LEUA 32 12.464 21.10021.2691.0040.32
ATOM164 CA LEUA 32 12.638 22.48320.8461.0040.10
ATOM165 CB LEUA 32 11.301 23.06820.3671.0039.37
ATOM166 CG LEUA 32 11.349 24.31219.4621.0040.36
ATOM167 CD1LEUA 32 9.943 24.62918.9951.0039.47
ATOM168 CD2LEUA 32 11.946 25.52020.1871.0038.65
ATOM169 C LEUA 32 13.205 23.35121.9581.0040.12
ATOM170 0 LEUA 32 14.023 24.23721.7021.0042.39
ATOM171 N ALAA 33 12.767 23.10223.1901.0039.67
ATOM172 CA ALAA 33 13.225 23.87724.3.461.0039.26
ATOM173 CB ALAA 33 12.600 23.32625.6301.0037.33
ATOM174 C ALAA 33 14.746 23.91424.4811.0038.24
ATOM175 0 ALAA 33 15.317 24.93924.8661.0039.05
ATOM176 N ASPA 34 15.400 22.79924.1'701.0039.56
ATOM177 CA ASPA 34 16.857 22.72324.2581.0040.96
ATOM178 CB ASPA 34 17.352 21.30023.9'761.0040.20
ATOM179 CG ASPA 34 17.006 20.32725.0831.0038.93
ATOM180 OD1ASPA 34 16.981 20.74226.2621.0041.79
ATOM181 OD2ASPA 34 16.777 19.14024.7781.0037.45
ATOM182 C ASPA 34 17.570 23.67223.3011.0042.49
ATOM183 0 ASPA 34 18.752 23.96223.41321.0044.27
ATOM184 N HISA 35 16.859 24.16822.2951.0042.65
ATOM185 CA HISA 35 17.477 25.04021.3:L01.0043.28
ATOM186 CB HISA 35 17.078 24.57019.9:L11.0043.83
ATOM187 CG HISA 35 17.309 23.10819.6911.0043.04
ATOM188 CD2HISA 35 16.455 22.05619.7231.0044.56
ATOM189 NDlHISA 35 18.563 22.57219.4921.0044.90
ATOM190 CE1HISA 35 18.472 21.25619.4_:_51.0044.90
ATOM191 NE2HISA 35 17.201 20.91819.5:141. 42.77
CO
ATOM192 C HISA 35 17.175 26.51921.4781.0044.83
ATOM193 0 HISA 35 18.097 27.33021.5671.0044.27
ATOM194 N ILEA 36 15.895 26.87821.5231.0045.92
ATOM195 CA ILEA 36 15.529 28.28321.6761.0047.35
ATOM196 CB ILEA 36 14.513 28.70920.5711.0049.18
ATOM197 CG2ILEA 36 13.106 28.84721.1431.0049.20
ATOM198 CGlILEA 36 14.986 30.01419.9211.0049.48
ATOM199 CD1ILEA 36 15.256 31.13620.9021.0051.35
ATOM200 C ILEA 36 14.989 28.62223.0731.0046.83
ATOM201 0 ILEA 36 14.593 29.75623.3451.0046.83
ATOM202 N GLYA 37 14.993 27.63923.9Fi61.0046.04
ATOM203 CA GLYA 37 14.511 27.88525.37.61.0045.48
ATOM204 C GLYA 37 13.082 27.43025.5531.0043.62
ATOM205 O GLYA 37 12.346 27.15124.67.01.0043.67
ATOM206 N PHEA 38 12.694 27.36026.8211.0042.00
ATOM207 CA PHEA 38 11.354 26.93227.2001.0042.44
ATOM208 CB PHEA 38 11.356 26.49228.6761.0042.09
ATOM209 CG PHEA 38 10.116 26.87529.4271.0043.59
ATOM210 CDlPHEA 38 8.890 26.28429.1~s61.0042.94
ATOM211 CD2PHEA 38 10.167 27.86930.4001.0044.79
ATOM212 CE1PHEA 38 7.730 26.68529.7971.0042.28
ATOM213 CE2PHEA 38 9.016 28.27531.0631.0044.67
ATOM214 CZ PHEA 38 7.795 27.68030.7601.0043.27
ATOM215 C PHEA 38 10.266 27.98626.9501.0041.79
ATOM216 O PHEA 38 9.248 27.68526.3261.0040.84
ATOM217 N GLNA 39 10.475 29.21327.4181.0042.13
ATOM218 CA GLNA 39 9.472 30.26627.2921.0044.46
ATOM219 CB GLNA 39 9.880 31.54327.9851.0046.30
ATOM220 CG GLNA 39 10.027 31.36929.4881.0048.51
ATOM221 CD GLNA 39 10.066 32.69330.2291.0050.88
7
CA 02446527 2003-10-23
ATOM222 OE1GLNA 39 9.079 33.43430.2511.0050.83
ATOM223 NE2GLNA 39 11.208 32.99830.8431.0051.78
ATOM224 C GLNA 39 9.177 30.60725.7861.0045.01
ATOM225 O GLNA 39 8.075 31.04825.4571.0045.18
ATOM226 N GLUA 40 10.155 30.40724.9:L21.0045.57
ATOM227 CA GLUA 40 9.955 30.70023.5001.0044.79
ATOM228 CB GLUA 40 11.281 31.09622.8461.0046.36
ATOM229 CG GLUA 40 11.131 31.64721.4381.0048.30
ATOM230 CD GLUA 40 10.174 32.82021.3'741.0049.05
ATOM231 OE1GLUA 40 10.392 33.80422.1161.0050.84
ATOM232 OE2GLUA 40 9.204 32.75520.5831.0048.60
ATOM233 C GLUA 40 9.368 29.47522.8081.0043.43
ATOM234 0 GLUA 40 8.620 29.59221.8331.0042.89
ATOM235 N ALAA 41 9.702 28.29923.3271.0041.16
ATOM236 CA ALAA 41 9.201 27.05122.7651.0039.01
ATOM237 CB ALAA 41 10.012 25.87723.2'751.0037.48
ATOM238 C ALAA 41 7.740 26.87723.1461.0038.28
ATOM239 0 ALAA 41 6.915 26.48522.3:L71.0038.27
ATOM240 N TYRA 42 7.422 27.16324.4()61.0036.31
ATOM241 CA TYRA 42 6.048 27.04124.8801.0034.56
ATOM242 CB TYRA 42 5.937 27.49726.3401.0032.46
ATOM243 CG TYRA 42 4.592 27.19726.9801.0029.55
ATOM244 CD1TYRA 42 4.343 25.96627.5911.0027.43
ATOM245 CE1TYRA 42 3.090 25.67328.1511.0027.03
ATOM246 CD2TYRA 42 3.561 28.13526.9501.0028.09
ATOM247 CE2TYRA 42 2.308 27.85227.5041.0028.26
ATOM248 CZ TYRA 42 2.082 26.62728.1031.0028.64
ATOM249 OH TYRA 42 0.843 26.36228.6461.0030.72
ATOM250 C TYRA 42 5.173 27.92323.9911:0036.12
ATOM251 O TYRA 42 4.152 27.47523.4'711.0036.61
ATOM252 N ARGA 43 5.591 29.17423.8:L31.0036.78
ATOM253 CA ARGA 43 4.863 30.12722.9831.0040.45
ATOM254 CB ARGA 43 5.614 31.46222.9181.0042.48
ATOM255 CG ARGA 43 5.062 32.40821.8651.0046.79
ATOM256 CD ARGA 43 6.001 33.55921.5651.0049.17
ATOM257 NE ARGA 43 5.896 33.96120.1661.0052.05
ATOM258 CZ ARGA 43 6.187 33.16319.1411. 53.09
CO
ATOM259 NHlARGA 43 6.603 31.92419.3631. 54.07
CO
ATOM260 NH2ARGA 43 6.056 33.59517.8921.0052.56
ATOM261 C ARGA 43 4.640 29.61021.5651.0041.08
ATOM262 0 ARGA 43 3.581 29.83320.9801.0040.43
ATOM263 N ARGA 44 5.643 28.92521.0171.0041.91
ATOM264 CA ARGA 44 5.566 28.38019.6681.0041.07
ATOM265 CB ARGA 44 6.915 27.78219.2_'i01.0045.64
ATOM266 CG ARGA 44 7.861 28.77718.5761.0048.95
ATOM267 CD ARGA 44 7.141 29.51317.4481.0053.17
ATOM268 NE ARGA 44 6.442 28.58516.5:191.0057.69
ATOM269 CZ ARGA 44 5.469 28.93515.72.01.0059.72
ATOM270 NH1ARGA 44 4.895 28.02014.9511.0060.60
ATOM271 NH2ARGA 44 5.061 30.19715.6551.0060.94
ATOM272 C ARGA 44 4.478 27.33419.4881.0040.12
ATOM273 O ARGA 44 3.787 27.33418.4761.0038.24
ATOM274 N PHEA 45 4.324 26.44620.4671.0039.96
ATOM275 CA PHEA 45 3.312 25.39320.3871.0038.49
ATOM276 CB PHEA 45 3.824 24.10121.0261.0040.88
ATOM277 CG PHEA 45 4.808 23.34220.1841.0043.51
ATOM278 CDlPHEA 45 6.129 23.75920.0821.0044.81
ATOM279 CD2PHEA 45 4.413 22.18819.51.31.0045.28
ATOM280 CElPHEA 45 7.051 23.03219.3271.0045.40
ATOM281 CE2PHEA 45 5.322 21.45518.7561.0046.39
ATOM282 CZ PHEA 45 6.648 21.87918.6641.0046.57
ATOM283 C PHEA 45 1.969 25.72121.0451.0036.87
ATOM284 0 PHEA 45 0.969 25.06520.7611.0036.85
ATOM285 N TYRA 46 1.935 26.71421.9271.0034.86
ATOM286 CA TYRA 46 0.694 27.02522.62.41.0033.03
ATOM287 CB TYRA 46 0.827 26.59824.0811.0029.21
ATOM288 CG TYRA 46 1.202 25.15424.2221.0028.43
ATOM289 CD1TYRA 46 2.446 24.77324.7~~61.0028.99
CA 02446527 2003-10-23
ATOM290 CE1 TYRA 46 2.790 23.42724.8601.0025.13
ATOM291 CD2 TYRA 46 0.321 24.15623.8241.0027.13
ATOM292 CE2 TYRA 46 0.656 22.82223.9:361.0027.50
ATOM293 CZ TYRA 46 1.888 22.46124.4571.0026.34
ATOM294 OH TYRA a_6 2.174 21.12524.6:131.0027.54
ATOM295 C TYRA 46 0.240 28.47222.5521.0034.15
ATOM296 0 TYRA 46 -0.939 28.76622.7491.0035.46
ATOM297 N GLYA 47 1.170 29.37822.2781.0033.49
ATOM298 CA GLYA 47 0.802 30.77322.1891. 33.72
U0
ATOM299 C GLYA 47 -0.027 31.05920.9511.0036.70
ATOM300 0 GLYA 47 -0.146 30.22920.0441.0034.81
ATOM301 N ILEA 48 -0.634 32.23720.9251.0037.59
ATOM302 CA ILEA 48 -1.421 32.64719.7831.0040.72
ATOM303 CB ILEA 48 -2.751 33.29520.2021.0040.52
ATOM304 CG2 ILEA 48 -3.465 33.86618.9'721.0039.46
ATOM305 CG1 ILEA 48 -3.629 32.26020.9051.0040.08
ATOM306 CDl ILEA 48 -4.898 32.83921.4931.0041.90
ATOM307 C ILEA 48 -0.549 33.67919.1021.0043.37
ATOM308 O ILEA 48 -0.447 34.81719.5611.0044.91
ATOM309 N ALAA 49 0.100 33.26218.0211.0045.31
ATOM310 CA ALAA 49 0.999 34.13017.2061.0047.31
ATOM311 CB ALAA 49 0.427 35.55117.1'791.0048.09
ATOM312 C ALAA 49 2.381 34.15217.9211.0046.55
ATOM313 0 ALAA 49 2.587 33.37418.8811.0045.60
ATOM314 OXT ALAA 49 3.237 34.93817.4621.0045.24
ATOM315 CA+2CA2A 1 13.077 17.43332.2'711.0022.23
C
ATOM316 CA+2CA2A 2 13.835 18.86728.8371.0030.50
C
ATOM317 CA+2CA2A 3 10.897 18.81335.3351.0050.79
C
ATOM318 OH2 TIPA 1 5.850 30.87628.8'751.0026.66
S
ATOM319 OH2 TIPA 2 13.387 22.46133.5301.0024.93
S
ATOM320 OH2 TIPA 3 2.021 19.16026.9'!91.0035.80
S
ATOM321 OH2 TIPA 4 5.803 14.66619.0''11.0038.16
S
ATOM322 OH2 TIPA 5 10.578 15.30429.5671.0023.15
S
ATOM323 OH2 TIPA 6 5.020 20.56340.6361.0044.02
S
ATOM324 OH2 TIPA 7 2.823 22.14438.5~L61.0036.74
S
ATOM325 OH2 TIPA 8 10.434 22.63129.6041.0025.89
S
ATOM326 OH2 TIPA 9 6.522 15.69136.4731.0027.82
S
ATOM327 OH2 TIPA 10 2.927 29.39538.6491.0033.09
S
ATOM328 OH2 TIPA 11 8.208 35.76532.3381.0047.23
S
ATOM329 OH2 TIPA 12 14.353 36.47034.8201.0066.90
S
ATOM330 OH2 TIPA 13 3.807 28.48234.8241.0024.88
S
ATOM331 OH2 TIPA 14 11.624 15.35831.8221.0024.92
S
ATOM332 OH2 TIPA 15 13.763 16.79828.6671.0029.47
S
ATOM333 OH2 TIPA 16 6.350 16.97332.3~L01.0037.83
S
ATOM334 OH2 TIPA 17 9.425 33.46443.0951.0058.60
S
ATOM335 OH2 TIPA 18 3.199 34.74428.5391.0035.94
S
ATOM336 OH2 TIPA 19 13.597 33.84746.4(i71.0045.84
S
ATOM337 OH2 TIPA 20 10.474 21.05434.7391.0025.48
S
ATOM338 OH2 TIPA 21 8.008 14.32126.2701.0036.62
S
ATOM339 OH2 TIPA 22 5.694 31.58326.4131.0054.83
S
ATOM340 OH2 TIPA 23 6.216 35.11327.4491.0038.82
S
ATOM341 OH2 TIPA 24 16.203 18.68827.7201.0028.10
S
ATOM342 OH2 TIPA 25 8.186 14.32730.4771.0049.44
S
ATOM343 OH2 TIPA 26 8.625 16.47733.8Ei81.0048.13
S
ATOM344 OH2 TIPA 27 5.125 11.77028.0381.0039.55
S
ATOM345 OH2 TIPA 28 2.083 16.11925.7811.0038.18
S
ATOM346 OH2 TIPA 29 15.462 16.71424.7891.0042.90
S
ATOM347 OH2 TIPA 30 13.510 28.01631.3381.0058.36
S
ATOM348 OH2 TIPA 31 3.415 31.46425.2711.0046.78
S
ATOM349 OH2 TIPA 33 -0.797 33.53523.5391.0027.00
S
ATOM350 OH2 TIPA 34 -1.094 29.63425.8051.0039.49
S
ATOM351 OH2 TIPA 35 1.137 31.11126.31.01.0031.80
S
ATOM352 OH2 TIPA 36 1.407 37.00128.2101.0037.32
S
ATOM353 OH2 TIPA 37 0.970 33.42527.5201.0052.33
S
ATOM354 OH2 TIPA 38 -2.315 31.72329.9531.0044.23
S
ATOM355 OH2 TIPA 39 4.757 17.42338.8791.0034.26
S
ATOM356 OH2 TIPA 4C 3.611 36.97827.0271.0033.99
S
ATOM357 OH2 TIPA 41 10.313 14.49525.4521.0040.66
S
CA 02446527 2003-10-23
ATOM358 OH2TIPA 42 1.979 18.61637.7601.0034.25
S
ATOM359 OH2TIPA 43 5.964 18.90916.4121.0039.77
S
ATOM360 OH2TIPA 44 1.860 21.46134.6'731.0032.95
S
ATOM361 OH2TIPA 45 11.462 18.11317.4611.0052.62
S
ATOM362 OH2TIPA 46 13.926 X .62727.2711.0029.62
S
ATOM363 OH2TIPA 47 19.590 28.29919.0781.0043.97
S
ATOM364 OH2TIPA 48 16.240 23.47127.7001.0048.79
S
ATOM365 OH2TIPA 49 4.036 16.71434.0841.0048.66
S
ATOM366 OH2TIPA 50 12.966 33.07518.81 1.0057.37
6 S
ATOM367 OH2TIPA 51 4.126 14.41736.3411.0040.73
S
ATOM368 OH2TIPA 52 11.703 37.54330.6511.0036.00
S
ATOM369 OH2TIPA 53 2.747 18.82335.1701.0050.30
S
ATOM370 OH2TIPA 54 0.279 24.29338.8991.0043.36
S
ATOM371 OH2TIPA 55 5.228 23.55341.5591.0042.02
S
ATOM372 OH2TIPA 56 5.298 21.83343.4'731.0041.96
S
ATOM373 OH2TIPA 57 -2.985 34.43224.6881.0037.28
S
ATOM374 OH2TIPA 58 9.768 32.88636.7=~51.0030.34
S
ATOM375 OH2TIPA 59 11.644 31.77915.2091.0038.45
S
ATOM376 OH2TIPA 60 13.181 22.61329.2.L01.0035.43
S
ATOM377 OH2TIPA 63 3.510 13.29933.0521.0044.76
S
ATOM378 OH2TIPA 64 23.246 30.85339.7771.0053.00
S
TER
END
l
