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PDBsum entry 1tq0
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Blood clotting
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PDB id
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1tq0
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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The anticoagulant thrombin mutant w215a/e217a has a collapsed primary specificity pocket.
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Authors
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A.O.Pineda,
Z.W.Chen,
S.Caccia,
A.M.Cantwell,
S.N.Savvides,
G.Waksman,
F.S.Mathews,
E.Di cera.
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Ref.
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J Biol Chem, 2004,
279,
39824-39828.
[DOI no: ]
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PubMed id
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Abstract
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The thrombin mutant W215A/E217A features a drastically impaired catalytic
activity toward chromogenic and natural substrates but efficiently activates the
anticoagulant protein C in the presence of thrombomodulin. As the remarkable
anticoagulant properties of this mutant continue to be unraveled in preclinical
studies, we solved the x-ray crystal structures of its free form and its complex
with the active site inhibitor H-d-Phe-Pro-Arg-CH(2)Cl (PPACK). The PPACK-bound
structure of W215A/E217A is identical to the structure of the PPACK-bound slow
form of thrombin. On the other hand, the structure of the free form reveals a
collapse of the 215-217 strand that crushes the primary specificity pocket. The
collapse results from abrogation of the stacking interaction between Phe-227 and
Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other
notable changes are a rotation of the carboxylate group of Asp-189, breakage of
the H-bond between the catalytic residues Ser-195 and His-57, breakage of the
ion pair between Asp-222 and Arg-187, and significant disorder in the 186- and
220-loops that define the Na(+) site. These findings explain the impaired
catalytic activity of W215A/E217A and demonstrate that the analysis of the
molecular basis of substrate recognition by thrombin and other proteases
requires crystallization of both the free and bound forms of the enzyme.
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Figure 2.
FIG. 2. Stereo view of the active site, primary specificity
pocket, and Na^+-binding site of the thrombin mutant WE. The
PPACK-inhibited WE structure (blue) is superimposed to the SL
structure (red) of the wild type (15). Notwithstanding the
drastic difference in atomic resolution (2.4 Å for
WE-PPACK and 1.55 Å for SL), the two structures are
remarkably similar overall (r.m.s. deviation = 0.4 Å).
There is no evidence of bound Na^+ in the WE-PPACK structure,
and there is a notable 1:1 correspondence for the water
molecules in the Na^+ site between the two structures. Relevant
side chains are labeled. In the WE structure, the side chain of
Lys-224 moves away from residue 217 because of the E217A
mutation.
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Figure 3.
FIG. 3. Stereo view of the active site and primary
specificity pocket of the thrombin mutant WE. The free form of
WE (red), shown with the 2F[o] - F[c] electron density map
contoured at 0.7  level (orange), is
superimposed to the PPACK-inhibited form (blue). The 215-217
strand in the free form collapses into the primary specificity
pocket and clashes with the Arg residue at the P1 position of
PPACK (green). The r.m.s. deviation between free WE and WE-PPACK
in the 215-221 segment is 2.5 Å. The r.m.s. deviation
between the two monomers in the asymmetric unit of the free WE
structure in the same segment is 0.5 Å. Also notable is
the rotation of the side chain of Asp-189 in the free form that
aligns almost parallel to the backbone as well as the shift in
the side chain of Ser-195 that moves away from its H-bonding
partner His-57.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
39824-39828)
copyright 2004.
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