Crystal Structure of the Anticoagulant Slow Form of Thrombin

Thrombin is the ultimate protease of the blood clotting cascade and plays a major role in its own regulation. The ability of thrombin to exhibit both pro-and anti-coagulant properties has spawned efforts to turn thrombin into an anticoagulant for therapeutic purposes. This quest culminated in the identification of the E217K variant through scanning and saturation mutagenesis. The antithrombotic properties of E217K thrombin are derived from its inability to convert fibrinogen to a fibrin clot while maintaining its thrombomodulin-dependent ability to activate the anticoagulant protein C pathway. Here we describe the 2.5-Å crystal structure of human E217K thrombin, which displays a dramatic restructuring of the geometry of the active site. Of particular interest is the repositioning of Glu-192, which hydrogen bonds to the catalytic Ser-195 and which results in the complete occlusion of the active site and the destruction of the oxyanion hole. Substrate binding pockets are further blocked by residues previously implicated in thrombin allostery. We have concluded that the E217K mutation causes the allosteric inactivation of thrombin by destabilizing the Na ؉ binding site and that the structure thus may represent the Na ؉ -free, catalytically inert "slow" form.Thrombin activity is central to hemostasis, the balance between thrombosis and bleeding (for reviews, see Refs. 1, 2), and consequently thrombin is an important target of anticoagulant therapies (for review, see Ref.3). Thrombin is generated from its zymogen form, prothrombin, at the end of the coagulation cascade and is eventually inhibited by the circulating serpin antithrombin (for review, see Ref. 4). Thrombin has many procoagulant properties, including the cleavage of fibrinogen to fibrin, which then polymerizes to form the fibrin clot. Thrombin is also responsible for activating the transglutaminase factor XIII, which stabilizes the clot by cross-linking the fibrin polymers. Thrombin activates platelets by cleaving protease-activated receptors and stimulates its own generation by activating cofactors V and VIII. Hemostasis is dependent on limiting procoagulant activity to surfaces of the vasculature that have been compromised. Thus, when thrombin leaches away from the site of tissue damage its activity is reversed from pro-to anti-coagulant by binding to the integral membrane protein, thrombomodulin (TM), 1 expressed at the surface of the intact endothelium (5). Once bound to TM, thrombin can no longer cleave fibrinogen and instead cleaves protein C to yield activated protein C. Activated protein C then dampens thrombin generation by cleavage inactivation of cofactors Va and VIIIa (for review, see Ref. 6).Thrombin interacts with many cofactors capable of inducing conformational change and altered protease activity; however, the physiological significance of thrombin allostery is unclear (7-10). The most relevant alteration of thrombin activity is caused by its binding to TM, but similar changes can also be induced by other cofactors. Of particular interest...

Section: Fig 4 Conformational Changes In the Namentioning

confidence: 90%

Section: Fig 4 Conformational Changes In the Namentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Crystal Structure of Anticoagulant Thrombin Variant E217K Provides Insights into Thrombin Allostery

Carter

Myles

Gibbs

et al. 2004

“…Crystallization of Thrombin Mutants G193A and G193P-The thrombin mutants were expressed, purified, and tested for activity as described previously (15,16). The mutants were then concentrated to 5 mg/ml in 50 mM choline chloride, 20 mM MES, 1 pH 6.0.…”

Section: Expression and Purification Of Trypsin Mutants In Pichia Pasmentioning

confidence: 99%

Energetic and Structural Consequences of Perturbing Gly-193 in theOxyanion Hole of SerineProteases

Bobofchak

Pineda

Mathews

et al. 2005

Self Cite

The oxyanion hole of serine proteases is formed by the backbone N atoms of the catalytic Ser-195 and Gly-193 and engages the backbone O atom of the P1 residue of substrate in an important H-bonding interaction. The energetic contribution of this interaction in the ground and transition states is presently unknown. Measurements of the individual rate constants defining the catalytic mechanism of substrate hydrolysis for wild-type thrombin and trypsin and their G193A and G193P mutants reveal that Gly-193 is required for optimal substrate binding and acylation. Crystal structures of the G193A and G193P mutants of thrombin bound to the active site inhibitor H-D-Phe-Pro-Arg-CH 2 Cl document the extent of perturbation induced by the replacement of Gly-193. The Ala mutant weakens the H-bonding interaction of the N atom of residue 193, whereas the Pro substitution abrogates it altogether with additional small shifts of the protein backbone. From the kinetic and structural data, we estimate that the H-bonding interaction in the oxyanion hole contributes a stabilization of the ground and transition states of >1.5 kcal/mol but <3.0 kcal/mol. These results shed light on a basic aspect of the enzyme-substrate interaction in the entire family of trypsin-like serine proteases.Serine proteases catalyze the hydrolysis of peptide bonds using the triad His-57/Asp-102/Ser-195 (chymotrypsinogen numbering) (1, 2). Binding of substrate to the active site is stabilized by a network of H-bonds, five of which are highly conserved and ensure the efficient hydrolysis of amide bonds. Of these H-bonds, two stabilize an anti-parallel ␤-sheet between Gly-216 and the P3 residue of substrate, whereas the other three involve the backbone N and O atoms of the P1 residue of substrate. The N atom engages the backbone O atom of Ser-214, often referred to as the "fourth" member of the catalytic machinery (3). The O atom of the P1 residue, on the other hand, makes two H-bonding interactions with the backbone N atoms of the catalytic in the so-called "oxyanion hole." The role of this region of the enzyme is to stabilize the developing negative charge on the O atom of substrate during formation of the tetrahedral intermediate (4). The lack of a side chain at position 193 allows correct positioning of the amido hydrogen to form the requisite H-bond with the oxyanion substrate. Indeed, Gly is highly conserved at position 193 in serine proteases, but few exceptions do exist and are associated with perturbed substrate hydrolysis and resistance to inhibition (5-8).Previous studies have addressed the important question of the energetic involvement of the oxyanion hole in substrate recognition. In subtlisin, one of the H-bonding group in the oxyanion hole is provided by the side chain of Asn-155 (9). Mutation of Asn-155 to the isosteric Leu, devoid of H-bonding capabilities, produces a derivative with reduced k cat /K m due to a significant (200-fold) decrease of k cat (9, 10). The absence of effect on K m has been interpreted as supporting the role of the H-b...

“…The inactive S* form was found incapable of binding Na ϩ and substrate at the active site (10). Following the pioneering structure of thrombin free of inhibitors and Na ϩ portraying the active slow form (9,11), a number of crystal structures of free thrombin have been obtained with the enzyme in various inactive forms (12)(13)(14)(15)(16)(17). Some of these structures (13,16,17) have been claimed to capture the essential properties of the inactive slow form S*, with the active site occluded and the Na ϩ site disordered.…”

mentioning

confidence: 99%

Crystal Structure of Thrombin in a Self-inhibited Conformation

Pineda

Chen

Bah

et al. 2006

Self Cite

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.