Allosteric activation of antithrombin is independent of charge neutralization or reversal in the heparin binding site.

We investigate the hypothesis that heparin activates antithrombin (AT) by relieving electrostatic strain within helix D. Mutation of residues K125 and R129 to either Ala or Glu abrogated heparin binding, but did not activate AT towards inhibition of factors IXa or Xa. However, substitution of residues C-terminal to helix D (R132 and K133) to Ala had minimal effect on heparin affinity but resulted in appreciable activation. We conclude that charge neutralization or reversal in the heparin binding site does not drive the activating conformational change of AT, and that the role of helix D elongation is to stabilize the activated state.

Expression and characterization of full-length human huntingtin, an elongated HEAT repeat protein.

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.

Crystal structure of thrombin bound to heparin.

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-A structure of human alpha-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.

Crystal structure of anticoagulant thrombin variant E217K provides insights into thrombin allostery.

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-A 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.