Crystal Structure of Thrombin in a Self-inhibited Conformation

Abstract

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.

Role of the A chain in thrombin function

“…The results have been interpreted in terms of molecular dynamics calculations as a gross perturbation of the active site moiety propagating long-range from the site of deletion in the A chain [9]. Notably, the perturbed structure produced by modeling contains features documented in the X-ray crystal structure of the inactive form of thrombin E* [25]. Recent measurements of the pKa of the catalytic His57 further support a direct effect on active site residues [10].…”

Structural identification of the pathway of long-range communication in an allosteric enzyme

“…However, the structure documents a large conformational change that propagates from Phe-34 and Arg-73 in exosite I to Trp-215 in the aryl binding site and Arg-221a in the 220-loop located up to 28-Å away on the opposite side of the active site relative to exosite I. The conformational change is revealed by comparison of the structure of D102N bound to the fragment of PAR1 at exosite I with that of the free mutant solved recently (42). The free mutant D102N folds in a self-inhibited conformation with the active site occluded (Fig.…”

“…Further downstream from Trp-215, Arg-221a relinquishes its ion-pair interaction with Glu-146 in the autolysis loop and penetrates the 220-loop to engage the carboxylate of Asp-189 in the primary specificity pocket with its guanidinium group. The drastic rearrangement of the 215-219 ␤-strand and the 220-loop shift the orientation of the Cys-191:Cys-220 disulfide bond and propagate the perturbation up to the oxyanion hole where the backbone N atom of Gly-193 is flipped relative to wild type (42). Binding of PAR1 to exosite I reverses all of these drastic changes and shifts the conformation of D102N into that of wild-type thrombin (21, 41) using a long-range allosteric communication between exosite I and the active site.…”

“…Human thrombin inactivated with the single-site mutation D102N was crystallized in complex with the extracellular fragment of human PAR1, 42 SFLLRNPNDKYEPFWEDEEKN 62 , corresponding to the sequence downstream from the cleavage site at Arg-41 (38) and containing the hirudin-like motif 52 YEPFWE 57 predicted to bind to exosite I (33,38). Although the structure was solved at a resolution of 2.2 Å, only the sequence 49 NDKYEPFWE 57 of the extracellular fragment of PAR1 could be traced in the electron density map, leaving the tethered ligand domain 42 SFLLRN 47 and the acidic tail of the fragment unresolved (Fig.…”

“…Similarly, the E*3E transition affects the structure of the enzyme as a whole (12,19). If E* is indeed an inactive conformation of thrombin (47,52), then the recent structure of the mutant D102N in the free form offers a possible representation (42). Given the current structural assignments of E*, E, and E:Na ϩ , one can conclude that the structural changes in the 60-loop documented upon PAR3 binding to exosite I of murine thrombin (32) pertain to a conformation of the enzyme that is stabilized in the highly active E:Na ϩ form (53,54).…”