Thrombin is the final protease in the blood coagulation cascade and serves both pro-and anticoagulant functions through the cleavage of several targets. The ability of thrombin to specifically recognize a wide range of substrates derives from interactions that occur outside of the active site of thrombin. Thrombin possesses two anion binding exosites, which mediate many of its interactions with cofactors and substrates, and although many structures of thrombin have been solved, few such interactions have been described in molecular detail. Glycosaminoglycan binding to exosite II of thrombin plays a major role in switching off the procoagulant functions of thrombin by mediating its irreversible inhibition by circulating serpins and by its binding to the endothelial cell surface receptor thrombomodulin. Here we report the 1.85-Å structure of human ␣-thrombin bound to a heparin fragment of eight monosaccharide units in length. The asymmetric unit is composed of two thrombin dimers, each sharing a single heparin octasaccharide chain. The observed interactions are fully consistent with previous mutagenesis studies and illustrate on a molecular level the cofactor interaction that is critical for the restriction of clotting to the site of blood vessel injury.Thrombin is the ultimate protease in the blood clotting cascade and is uniquely able to cleave soluble fibrinogen into polymerogenic fibrin, resulting in the formation of the fibrin clot. Thrombin also serves a critical role in stimulating its own generation from the zymogen prothrombin by cleavage activation of upstream coagulation factors and platelet receptors through positive feedback loops (for reviews, see Refs. 2-5). The procoagulant functions of thrombin are counterbalanced by the plasma serpins antithrombin (AT) 1 and heparin cofactor II (HCII) (for reviews, see Refs. 6 and 7) and by the integral membrane protein thrombomodulin (TM) (for review, see Refs. 8 and 9). AT and HCII inhibit thrombin in an irreversible fashion common to all members of the serpin family, whereas TM binding serves to alter the specificity of thrombin away from the prothrombotic substrates in favor of the anticoagulant protein C. Thus thrombin specificity is of paramount importance in determining the hemostatic balance between clotting and bleeding.Close examination of the peptide sequences comprising the natural substrates of thrombin provides little insight into how thrombin specificity is determined, other than to permit drawing the conclusion that determinants of thrombin substrate specificity lie elsewhere (3, 10). The solution of the crystallographic structure of thrombin revealed two cationic patches on the surface of thrombin denoted anion binding exosites I and II (1). Exosite I, on the eastern face of thrombin in the standard orientation (11), is adjacent to, and contiguous with, the active site cleft and is known to be the fibrinogen recognition exosite. Exosite II, on the northwestern face of thrombin, is the more basic of the two exosites and was identified as the putat...

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...

Huntington disease is an inherited neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats, resulting in a polyglutamine stretch of >37 on the N terminus of the protein huntingtin (htt). htt is a large (347 kDa), ubiquitously expressed protein.The precise functions of htt are not clear, but its importance is underscored by the embryonic lethal phenotype in htt knock-out mice. Despite the fact that the htt gene was cloned 13 years ago, little is known about the properties of the full-length protein. Here we report the expression and preliminary characterization of recombinant full-length wild-type human htt. Our results support a model of htt composed entirely of HEAT repeats that stack to form an elongated superhelix. Huntington disease (HD)3 is an autosomal-dominant neurodegenerative disorder that is caused by expanded CAG trinucleotide repeats on the N terminus of the IT15 gene that encodes the protein huntingtin (htt) (1). HD occurs in individuals whose htt gene has more than 37 CAGs, resulting in a mutant protein with an abnormally extended polyglutamine (polyQ) tract. Symptoms can manifest at any age and typically involve movement disorders, including chorea, along with psychiatric and cognitive dysfunction. The median age at onset is 40 years, with death generally following 15-20 years after appearance of symptoms (2, 3). Although the neurodegeneration caused by the HD mutation is particularly marked in the striatum and cortex, htt is widely expressed in many different tissues and its functions are critical for life (4 -6). Mutant htt is prone to aggregation, and both cytosolic and nuclear inclusions have been observed (7-10).Extensive genetic and transgenic data suggest that the HD mutation causes disease primarily by conferring a toxic gain-of-function on the mutant protein (Ref. 4 and reviewed in Ref. 11). However, it is possible that loss-of-function and/or dominant negative effects may also contribute to pathology (reviewed in Refs. 11,12). Proteolytic processing of htt is also likely to play an important role in HD pathogenesis. The toxicity of mutant htt may only be fully exposed after cleavage by proteases, including caspases, calpains, and a putative aspartic protease, to reveal a short, N-terminal polyQ-containing fragment of 100 -150 residues (reviewed in Refs. 13-15 and references therein). N-terminal cleavage products have been found in inclusions from HD patients (16 -18), and N-terminal fragments with expanded polyQs readily form inclusions similar to those seen in HD patients. However, it is still unclear which combination of proteolytic events is required for generation of the toxic fragments.There are nine different inherited neurodegenerative diseases caused by CAG/polyQ expansion in the target proteins. Although all show inclusions containing the expanded polyQs, the parts of the brain affected by the different polyQ-expanded proteins differ, resulting in different symptom constellations. Therefore, the host protein itself and its distinct interactions with other...