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PDBsum entry 3hk6
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References listed in PDB file
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Key reference
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Title
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Mechanism of the anticoagulant activity of thrombin mutant w215a/e217a.
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Authors
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P.S.Gandhi,
M.J.Page,
Z.Chen,
L.Bush-Pelc,
E.Di cera.
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Ref.
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J Biol Chem, 2009,
284,
24098-24105.
[DOI no: ]
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PubMed id
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Note: In the PDB file this reference is
annotated as "TO BE PUBLISHED". The citation details given above have
been manually determined.
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Abstract
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The thrombin mutant W215A/E217A (WE) is a potent anticoagulant both in vitro and
in vivo. Previous x-ray structural studies have shown that WE assumes a
partially collapsed conformation that is similar to the inactive E* form, which
explains its drastically reduced activity toward substrate. Whether this
collapsed conformation is genuine, rather than the result of crystal packing or
the mutation introduced in the critical 215-217 beta-strand, and whether binding
of thrombomodulin to exosite I can allosterically shift the E* form to the
active E form to restore activity toward protein C are issues of considerable
mechanistic importance to improve the design of an anticoagulant thrombin mutant
for therapeutic applications. Here we present four crystal structures of WE in
the human and murine forms that confirm the collapsed conformation reported
previously under different experimental conditions and crystal packing. We also
present structures of human and murine WE bound to exosite I with a fragment of
the platelet receptor PAR1, which is unable to shift WE to the E form. These
structural findings, along with kinetic and calorimetry data, indicate that WE
is strongly stabilized in the E* form and explain why binding of ligands to
exosite I has only a modest effect on the E*-E equilibrium for this mutant. The
E* --> E transition requires the combined binding of thrombomodulin and
protein C and restores activity of the mutant WE in the anticoagulant pathway.
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Figure 1.
Cα traces of engineered proteases hWE and mWE compared with
native thrombin. Left, new hWE structure with only one molecule
in the asymmetric unit (wheat) is nearly identical to the
previous structure, 1TQ0 (light green), and differs from the
active E form (1SGT (red)) (48) for the collapse of the
215–217 β-strand (arrow) into the active site. Right, mWE-1
(light blue), mWE-2 (wheat), and mWE-3 (light green) differ from
wild-type murine thrombin (2OCV (red)) (51) at both the
215–217 β-strand (arrow) and the oxyanion hole.
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Figure 4.
PAR1 binding to hWE and mWE does not restore active site
architecture. Left, hWE free (wheat) and hWE bound (light green)
to the PAR1 peptide (gold) are nearly identical with the
exception of the oxyanion hole (see legend for Fig. 3). Right,
mWE free (wheat) is nearly identical to the PAR1 (gold) bound
state (light green).
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2009,
284,
24098-24105)
copyright 2009.
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Secondary reference #1
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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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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
reproduced from the cited reference
with permission from the ASBMB
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