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J. Biol. Chem., Vol. 279, Issue 25, 26387-26394, June 18, 2004

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Crystal Structure of Anticoagulant Thrombin Variant E217K Provides Insights into Thrombin Allostery^*

Wendy J. Carter, Timothy Myles, Craig S. Gibbs¶, Lawrence L. Leung, and James A. Huntington||

From the University of Cambridge, Department of Haematology, Division of Structural Medicine, Thrombosis Research Unit, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom, the Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, California 94305, and ^¶Gilead Sciences, Foster City, California 94404

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-Å 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.

Received for publication, March 2, 2004 , and in revised form, April 7, 2004.

The atomic coordinates and structure factors (code IRD3) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by National Institutes of Health Grants R01 HL68629 (to J. A. H) and R01 HL57530 (to L. L. L.) and by the Medical Research Council, UK. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains a supplementary video.

^|| To whom correspondence should be addressed. Tel.: 44-1223-763230; Fax: 44-1223-336827; E-mail: jah52{at}cam.ac.uk.

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