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

The solution structure of the isolated N-terminal fragment of streptococcal protein-G B1 domain has been investigated in H2O and TFE/H2O solution by CD and NMR to gain insight into the possible role that native beta-hairpin secondary structure elements may have in early protein folding steps. The fragment also has been studied under denaturing conditions (6 M urea), and the resulting NMR chemical shifts were used as a reference for the disordered state. On the basis of CD and NMR data, it is concluded that in aqueous solution the fragment is basically flexible, with two local low populated chain bends involving residues 8-9 and 14-15, respectively, in close agreement with secondary structure predictions, a structure that is different from the final folded state of that segment of the protein. The changes in the CD spectrum, the presence of several medium-range NOEs plus two long-range NOEs, and the sign of the H alpha conformational shifts reveal that the addition of TFE facilitates the formation of a set of transient beta-hairpins involving essentially the same residues that form the native beta-hairpin found in the final three-dimensional structure of the B1 domain. The stabilization of native-like structures by TFE is known to occur for helices, but, to our knowledge, this is the first time the stabilization of a native-like beta-hairpin structure by TFE is reported. Since long-range tertiary interactions are absent in the isolated fragment, our results support the idea that, in addition to helices, beta-hairpins may play an active role in directing the protein folding process.

The activating effect of Na ؉ on thrombin is allosteric and depends on the conformational transition from a low activity Na ؉ -free (slow) form to a high activity Na ؉ -bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na ؉ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na ؉ site and the active site regions. Here, we report a 1.87 Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow 3 fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10 Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na ؉ -binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na ؉ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

The thrombin mutant W215A/E217A features a drastically impaired catalytic activity toward chromogenic and natural substrates but efficiently activates the anticoagulant protein C in the presence of thrombomodulin. As the remarkable anticoagulant properties of this mutant continue to be unraveled in preclinical studies, we solved the x-ray crystal structures of its free form and its complex with the active site inhibitor H-D-PhePro-Arg-CH 2 Cl (PPACK). The PPACK-bound structure of W215A/E217A is identical to the structure of the PPACK-bound slow form of thrombin. On the other hand, the structure of the free form reveals a collapse of the 215-217 strand that crushes the primary specificity pocket. The collapse results from abrogation of the stacking interaction between Phe-227 and Trp-215 and the polar interactions of Glu-217 with Thr-172 and Lys-224. Other notable changes are a rotation of the carboxylate group of Asp-189, breakage of the H-bond between the catalytic residues Ser-195 and His-57, breakage of the ion pair between Asp-222 and Arg-187, and significant disorder in the 186-and 220-loops that define the Na ؉ site. These findings explain the impaired catalytic activity of W215A/E217A and demonstrate that the analysis of the molecular basis of substrate recognition by thrombin and other proteases requires crystallization of both the free and bound forms of the enzyme.Thrombin possesses a paradoxical combination of procoagulant and anticoagulant roles (1). The procoagulant role involves the cleavage of fibrinogen and the platelet receptor PAR1, leading, respectively, to fibrin polymerization and platelet aggregation (2). The anticoagulant role unfolds on interaction with thrombomodulin and activation of protein C that eventually inhibits thrombin generation (3). The dual role of thrombin has long raised interest in dissociating its procoagulant and anticoagulant activities (4, 5). These efforts have culminated in the design of anticoagulant thrombin mutants that are capable of activating protein C but show significantly reduced activity toward fibrinogen and PAR1 both in vitro and in vivo (6 -8).The mutant W215A/E217A (WE) 1 is by far the most potent anticoagulant thrombin engineered to date (8) and shows a safe and potent anticoagulant/antithrombotic profile in vivo (9). The mutant has drastically reduced (4 -5 orders of magnitude) catalytic activity toward all thrombin substrates, whether chromogenic or natural. However, in the presence of thrombomodulin, the activity of the mutant toward protein C is restored to a level comparable with that of the wild type (8). As a result, WE can circulate in the blood for hours, acting to increase the concentration of the anticoagulant-activated protein C without eliciting any significant fibrinogen clotting or platelet aggregation (9). Because of the importance of WE as a potential treatment for fatal or debilitating thrombo-occlusive events such as myocardial infarction and ischemic stroke, there is a compelling reason to characterize its structural properties. H...

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