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PDBsum entry 1oyh
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Blood clotting
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PDB id
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1oyh
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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 influence of hinge region residue glu-381 on antithrombin allostery and metastability.
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Authors
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D.J.Johnson,
J.A.Huntington.
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Ref.
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J Biol Chem, 2004,
279,
4913-4921.
[DOI no: ]
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PubMed id
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Abstract
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Antithrombin becomes an efficient inhibitor of factor Xa and thrombin by binding
a specific pentasaccharide sequence found on a small fraction of the heparan
sulfate proteoglycans lining the microvaculature. In the structure of native
antithrombin, the reactive center loop is restrained due to the insertion of its
hinge region into the main beta-sheet A, whereas in the heparin-activated state
the reactive center loop is freed from beta-sheet A. In both structures, hinge
region residue Glu-381 makes several stabilizing contacts. To determine the role
of these contacts in the allosteric mechanism of antithrombin activation, we
replaced Glu-381 with an alanine. This variant is less active toward its target
proteases than control antithrombin, due to a perturbation of the equilibrium
between the two forms, and to an increase in stoichiometry of inhibition.
Pentasaccharide binding affinity is reduced 4-fold due to an increase in the
off-rate. These data suggest that the main role of Glu-381 is to stabilize the
activated conformation. Stability studies also showed that the E381A variant is
resistant to continued insertion of its reactive center loop upon incubation at
50 degrees C, suggesting new stabilizing interactions in the native structure.
To test this hypothesis, and to aid in the interpretation of the kinetic data we
solved to 2.6 A the structure of the variant. We conclude that wild-type Glu-381
interactions stabilize the activated state and decreases the energy barrier to
full loop insertion.
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Figure 1.
FIG. 1. The induced-fit heparin binding mechanism of
antithrombin. a, antithrombin (ribbon diagram) interacts with
heparin (ball-and-stick) in an apparent three step, four state
mechanism. The native state is in the low activity and low
affinity conformation with its reactive center loop (yellow and
green) partially inserted into the main  -sheet A (red). The
specific heparin pentasaccharide interacts with antithrombin
primarily via helices A (green) and D (cyan). Although kinetic
data fit a model with only one weak-binding intermediate, I[1],
a recent crystallographic structure suggests that there may be
an additional intermediate that has undergone all conformational
changes save expulsion of the reactive center loop and helix D
elongation, I[2]. Full activation occurs only after expulsion of
the hinge region of the reactive center loop, and the closing of
-sheet A to the five
stranded form (Activated). b-e, stereo representations of the
main chain and selected side chains of hinge region and
surrounding residues reveal important interactions in several
crystal forms of antithrombin. b-e correspond to native (  -glycoform, 1e05 [PDB]
), pentasaccharide-activated (1e03 [PDB]
), S380C-fluorescein-derivatized (1dzh [PDB]
), and the heparin-bound intermediate I2 (1nq9 [PDB]
), respectively. In native AT (b) the P13 Glu (381) bridges
strands 3, 4, and 5A to helix F residues (Arg-197 and Glu-195)
through a series of salt-bridges and hydrogen bonds. In the
activated conformation (c) strands 3 and 5A have annealed with
P13 Glu interacting with strands 2 and 3A and helix F. A
fluorescein moiety at the P14 position (d) blocked the native
P13 interactions leading to an activated conformation in
solution. One of the major conformational responses to heparin
binding in the intermediate I[2] (e) is the repositioning of
Tyr-220 so that it no longer interacts with P13 Glu. Residue
numbers are given for some of the residues for clarity.
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Figure 5.
FIG. 5. Stereo representations of C traces of AT colored
according to root mean squared deviation from native AT. a,
the C trace of the P13 Ala
(ball) variant reveals surprisingly profound main chain
differences when compared with the -glycoform of AT.
Although perturbation of the hinge region might be expected, the
effect seems to have propagated to the heparin binding region.
To determine which conformational changes are significant, and
which reflect the flexible nature of the molecule, the structure
of the -glycoform compared with
the -form is shown in b.
The color scheme reflects the RMSD from light gray to red for
0-3 Å. The distance between Lys-220 and -139 C is shown
as a dashed green line, and is increases from 5.1 to 7.8 Å
in response to the E381A mutation.
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The above figures are
reprinted
by permission from the ASBMB:
J Biol Chem
(2004,
279,
4913-4921)
copyright 2004.
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