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J. Biol. Chem., Vol. 279, Issue 30, 31842-31853, July 23, 2004

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Molecular Dissection of Na⁺ Binding to Thrombin^*

Agustin O. Pineda, Christopher J. Carrell, Leslie A. Bush, Swati Prasad, Sonia Caccia, Zhi-Wei Chen, F. Scott Mathews, and Enrico Di Cera

From the Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110

Na⁺ binding near the primary specificity pocket of thrombin promotes the procoagulant, prothrombotic, and signaling functions of the enzyme. The effect is mediated allosterically by a communication between the Na⁺ site and regions involved in substrate recognition. Using a panel of 78 Ala mutants of thrombin, we have mapped the allosteric core of residues that are energetically linked to Na⁺ binding. These residues are Asp-189, Glu-217, Asp-222, and Tyr-225, all in close proximity to the bound Na⁺. Among these residues, Asp-189 shares with Asp-221 the important function of transducing Na⁺ binding into enhanced catalytic activity. None of the residues of exosite I, exosite II, or the 60-loop plays a significant role in Na⁺ binding and allosteric transduction. X-ray crystal structures of the Na⁺-free (slow) and Na⁺-bound (fast) forms of thrombin, free or bound to the active site inhibitor H-D-Phe-Pro-Arg-chloromethyl-ketone, document the conformational changes induced by Na⁺ binding. The slow fast transition results in formation of the Arg-187:Asp-222 ion pair, optimal orientation of Asp-189 and Ser-195 for substrate binding, and a significant shift of the side chain of Glu-192 linked to a rearrangement of the network of water molecules that connect the bound Na⁺ to Ser-195 in the active site. The changes in the water network and the allosteric core explain the thermodynamic signatures linked to Na⁺ binding and the mechanism of thrombin activation by Na⁺. The role of the water network uncovered in this study establishes a new paradigm for the allosteric regulation of thrombin and other Na⁺-activated enzymes involved in blood coagulation and the immune response.

Received for publication, February 17, 2004 , and in revised form, April 14, 2004.

The atomic coordinates and structure factors (codes 1SGI, 1SG8, 1SHH, and 1SFQ) 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 in part by National Institutes of Health Research Grants HL49413, HL58141, and HL73813. 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.

Recipient of a Fellowship from the American Heart Association.

To whom correspondence should be addressed. Tel.: 314-362-4185; Fax: 314-747-5354; E-mail: enrico{at}biochem.wustl.edu.

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