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PDBsum entry 1rd3
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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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Crystal structure of anticoagulant thrombin variant e217k provides insights into thrombin allostery.
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Authors
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W.J.Carter,
T.Myles,
C.S.Gibbs,
L.L.Leung,
J.A.Huntington.
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Ref.
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J Biol Chem, 2004,
279,
26387-26394.
[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 were identified by an automated
search of PubMed on title and author
names, giving a
percentage match of
94%.
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Abstract
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Thrombin is the ultimate protease of the blood clotting cascade and plays a
major role in its own regulation. The ability of thrombin to exhibit both pro-
and anti-coagulant properties has spawned efforts to turn thrombin into an
anticoagulant for therapeutic purposes. This quest culminated in the
identification of the E217K variant through scanning and saturation mutagenesis.
The antithrombotic properties of E217K thrombin are derived from its inability
to convert fibrinogen to a fibrin clot while maintaining its
thrombomodulin-dependent ability to activate the anticoagulant protein C
pathway. Here we describe the 2.5-A crystal structure of human E217K thrombin,
which displays a dramatic restructuring of the geometry of the active site. Of
particular interest is the repositioning of Glu-192, which hydrogen bonds to the
catalytic Ser-195 and which results in the complete occlusion of the active site
and the destruction of the oxyanion hole. Substrate binding pockets are further
blocked by residues previously implicated in thrombin allostery. We have
concluded that the E217K mutation causes the allosteric inactivation of thrombin
by destabilizing the Na(+) binding site and that the structure thus may
represent the Na(+)-free, catalytically inert "slow" form.
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Figure 2.
FIG. 2. E217K thrombin is inactive because of the altered
hydrogen bonding of the active site residues. a, stereo
representation of the electron density surrounding residues
His-57, Trp-60d, 191-196 and 215-220. The conformation of this
region is significantly altered for the E217K variant, and as a
result of the movement of Glu-192, the hydrogen bonding of the
catalytic residues has become non-catalytic. b, superposition of
the catalytic residues (His-57 and residues 192-195) of E217K
(yellow) and wild-type, active thrombin (1HAH [PDB]
, magenta) illustrates the new hydrogen bonding pattern (green
broken lines) and the consequent loss of the oxyanion hole,
normally formed by the amide hydrogens of Gly-193 and Ser-195
(arrows). The amide hydrogen of Gly-193 is oriented away from
the oxyanion hole, and the amide hydrogen of Ser-195 is
hydrogen-bonded to the main chain oxygen of Glu-192. The
hydrogens of Ser-195 are shown for clarity (white).
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Figure 3.
FIG. 3. Surface representations of thrombin reveal the
occlusion of the active site cleft caused by the E217K mutation.
a, the surface of active thrombin (1HAH [PDB]
, colored green for hydrophobicity) in the standard orientation
demonstrates the open active site cleft of thrombin and the
accessibility of the catalytic O  of Ser-195 (red). This
conformation represents an allosterically activated thrombin
with nothing bound in the active site cleft. However, the
structure can easily accommodate a natural thrombin substrate
derived from the reactive center loop of heparin cofactor II
(P4-P4', shown as rods). b, in contrast, the active site cleft
of E217K is in a closed conformation, and although the catalytic
O is colored as above, it
is fully blocked by the conformational changes in the active
site. In addition, it is clear that overlaps and steric clashes
from P4 to P1 would prevent substrate binding to E217K. A video
depiction of the structural transition from a closed (E217K) to
an open (1JOU [PDB]
, monomer AB) active site is given as supplementary material.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
26387-26394)
copyright 2004.
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