Molecular dissection of Na+ binding to thrombin

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 3 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.