The zymogen prothrombin is proteolytically converted by factor Xa to the active protease thrombin in a reaction that is accelerated >3,000-fold by cofactor Va. This physiologically important effect is paradigmatic of analogous cofactor-dependent reactions in the coagulation and complement cascades, but its structural determinants remain poorly understood. Prothrombin has three linkers connecting the N-terminal Gla domain to kringle-1 (Lnk1), the two kringles (Lnk2), and kringle-2 to the C-terminal protease domain (Lnk3). Recent developments indicate that the linkers, and particularly Lnk2, confer on the zymogen significant flexibility in solution and enable prothrombin to sample alternative conformations. The role of this flexibility in the context of prothrombin activation was tested with several deletions. Removal of Lnk2 in almost its entirety (ProTΔ146-167) drastically reduces the enhancement of thrombin generation by cofactor Va from >3,000-fold to 60-fold because of a significant increase in the rate of activation in the absence of cofactor. Deletion of Lnk2 mimics the action of cofactor Va and offers insights into how prothrombin is activated at the molecular level. The crystal structure of ProTΔ146-167 reveals a contorted architecture where the domains are not vertically stacked, kringle-1 comes within 9 Å of the protease domain, and the Gladomain primed for membrane binding comes in contact with kringle-2. These findings broaden our molecular understanding of a key reaction of the blood coagulation cascade where cofactor Va enhances activation of prothrombin by factor Xa by compressing Lnk2 and morphing prothrombin into a conformation similar to the structure of ProTΔ146-167.clotting factor | zymogen activation | structural biology T he clotting factor prothrombin is proteolytically converted by factor Xa to the active protease thrombin by cleavage at two sites, R271 and R320, along two mutually exclusive pathways producing the intermediates prethrombin-2 (cleavage at R271 first) or meizothrombin (cleavage at R320 first). The reaction is greatly accelerated through a drastic increase in k cat when catalyzed by the prothrombinase complex composed of factor Xa, cofactor Va, Ca 2+ , and phospholipids (1, 2). Both the rate enhancement and pathway of activation are under the control of cofactor Va and phospholipids (3, 4), although the cofactor has the greatest effect in enhancing k cat . The molecular determinants of this physiologically important effect are of substantial mechanistic interest because they bear on the cofactor-dependent activation of zymogens in many proteolytic cascades (5, 6).Prothrombin is composed of fragment 1 (residues 1-155), fragment 2 (residues 156-271), and a protease domain (residues 272-579). Fragment 1 contains the Gla domain (residues 1-46) and kringle-1 (residues 65-143), fragment 2 contains kringle-2 (residues 170-248), and the protease domain contains the A chain (residues 272-320) and the catalytic B chain (residues 321-579). Three linkers-to be referred to as Lnk1, Lnk2, ...

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 Å between the two forms, causing a displacement of 6.6 Å of the entire 215–217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.

Thrombin elicits functional responses critical to blood homeostasis by interacting with diverse physiological substrates. Ala-scanning mutagenesis of 97 residues covering 53% of the solvent accessible surface area of the enzyme identifies Trp 215 as the single most important determinant of thrombin specificity. Saturation mutagenesis of Trp 215 produces constructs featuring k cat /K m values for the hydrolysis of fibrinogen, protease-activated receptor PAR1, and protein C that span five orders of magnitude. Importantly, the effect of Trp 215 replacement is context dependent. Mutant W215E is 10-fold more specific for protein C than fibrinogen and PAR1, which represents a striking shift in specificity relative to wild-type that is 100-fold more specific for fibrinogen and PAR1 than protein C. However, when the W215E mutation is combined with deletion of nine residues in the autolysis loop, which by itself shifts the specificity of the enzyme from fibrinogen and PAR1 to protein C, the resulting construct features significant activity only toward PAR1. These findings demonstrate that thrombin can be re-engineered for selective specificity toward protein C and PAR1. Mutations of Trp 215 provide important reagents for dissecting the multiple functional roles of thrombin in the blood and for clinical applications.Engineering protease specificity remains an issue of considerable interest and importance (1). Within the same fold, trypsins prefer Arg/Lys side chains at the P1 position (2) of substrate but chymotrypsins prefer Phe/Tyr/Trp residues (3-6). Primary specificity, defined by the nature of the P1 residue, is not the sole determinant of protease function. Many trypsin-like proteases show substrate preference that extends beyond the P1 position of substrate and is dictated by interactions with other structural domains not in contact with the primary specificity pocket. Indeed, inspection of consensus sequences recognized by thrombin for its three primary physiological targets, i.e. the procoagulant substrate fibrinogen, the prothrombotic protease-activated receptor 1 (PAR1) 2 and the anticoagulant substrate protein C, reveals an Arg residue at the P1 position in all cases (7). Hence, selection among these targets must depend on interactions beyond the primary specificity pocket of the enzyme. A paradigm widely accepted in the field is that macromolecular specificity in thrombin and related clotting proteases is achieved and controlled by interaction with "exosites," i.e. domains widely separated from the active site region that kinetically control docking of substrate in ways that restrict choices by the enzyme (8, 9). However, abundant mutagenesis data show that residues within the active site play roles that far exceed in importance those of exosites and, in fact, affect specificity and the choice of which substrate can be cleaved by the enzyme in ways that are unmatched by other domains (10 -13). Consistent with these findings, the present study demonstrates that residue 215 within the thrombin active site is th...

The clotting factor prothrombin exists in equilibrium between closed and open conformations, but the physiological role of these forms remains unclear. As for other allosteric proteins, elucidation of the linkage between molecular transitions and function is facilitated by reagents stabilized in each of the alternative conformations. The open form of prothrombin has been characterized structurally, but little is known about the architecture of the closed form that predominates in solution under physiological conditions. Using X-ray crystallography and single-molecule FRET, we characterize a prothrombin construct locked in the closed conformation through an engineered disulfide bond. The construct: (i) provides structural validation of the intramolecular collapse of kringle-1 onto the protease domain reported recently; (ii) documents the critical role of the linker connecting kringle-1 to kringle-2 in stabilizing the closed form; and (iii) reveals novel mechanisms to shift the equilibrium toward the open conformation. Together with functional studies, our findings define the role of closed and open conformations in the conversion of prothrombin to thrombin and establish a molecular framework for prothrombin activation that rationalizes existing phenotypes associated with prothrombin mutations and points to new strategies for therapeutic intervention.

Prethrombin-2 is the immediate zymogen precursor of the clotting enzyme thrombin, which is generated upon cleavage at R15 and separation of the A chain and catalytic B chain. The X-ray structure of prethrombin-2 solved in the free form at 1.9 Å resolution shows the 215–217 segment collapsed into the active site. Remarkably, some of the crystals harvested from the same crystallization well diffract to 2.2 Å resolution in the same space group but produce a structure where the 215–217 segment does not hinder access to the active site. The two alternative conformations of prethrombin-2, open and collapsed, echo the active E and inactive E* forms of the mature enzyme. These findings validate the emerging paradigm that the allosteric E*-E equilibrium is a key property of the trypsin fold and demonstrate that the E and E* forms coexist under the same solution conditions and can be trapped by harvesting different crystals in the same crystallization well. Another unanticipated feature of prethrombin-2 is that R15 is buried within the protein in ionic interactions with E14e, D14l and E18 and its exposure is necessary for proteolytic attack and conversion to thrombin. Based on this structural observation, we constructed the E14eA/D14lA/E18A triple mutant to reduce electrostatic coupling with R15 and promote zymogen activation. The mutation causes prethrombin-2 to spontaneously convert to thrombin, without the need for the snake venom ecarin or the physiological prothrombinase complex.