Showing papers by "James A. Huntington published in 2005"

Crystal structure of wild-type human thrombin in the Na+-free state.

Abstract: Regulation of thrombin activity is critical for haemostasis and the prevention of thrombosis. Thrombin has several procoagulant substrates, including fibrinogen and platelet receptors, and essential cofactors for stimulating its own formation. However, thrombin is also capable of serving an anticoagulant function by activating protein C. The specificity of thrombin is primarily regulated by binding to the cofactor TM (thrombomodulin), but co-ordination of Na+ can also affect thrombin activity. The Na+-free form is often referred to as 'slow' because of reduced rates of cleavage of procoagulant substrates, but the slow form is still capable of rapid activation of protein C in the presence of TM. The molecular basis of the slow proteolytic activity of thrombin has remained elusive, in spite of two decades of solution studies and many published crystallographic structures. In the present paper, we report the first structure of wild-type unliganded human thrombin grown in the absence of co-ordinating Na+. The Na+-binding site is observed in a highly ordered position 6 A (1 A=0.1 nm) removed from that seen in the Na+-bound state. The movement of the Na+ loop results in non-catalytic hydrogen-bonding in the active site and blocking of the S1 and S2 substrate-binding pockets. Similar, if more dramatic, changes were observed in a previous structure of the constitutively slow thrombin variant E217K. The slow behaviour of thrombin in solutions devoid of Na+ can now be understood in terms of an equilibrium between an inert species, represented by the crystal structure described in the present paper, and an active form, where the addition of Na+ populates the active state.

Heparin Activation of Serpins

Abstract: Publisher Summary Serpins utilize a suicide substrate mechanism for the inhibition of serine proteases, which is best described by the kinetic model of protease substrate hydrolysis. The hallmark of heparin (HP) binding serpins is that the target protease inhibition is accelerated in the presence of HP. Acceleration is generally through a bridging mechanism where protease and serpin bind to the same HP chain, resulting in improved encounter rate and stabilization of Michaelis complex by reducing the initial dissociation rate. Antithrombin (AT) is the most important serpin in hemostasis. The importance of AT is demonstrated by the high association of deficiency with venous thrombosis, by the embryonic lethal consumptive coagulopathy in the knockout mouse, and by the continued success of therapeutic HP. HP acceleration of protease inhibition by AT involves two distinct mechanisms, depending on the targets involved. An HP binding site adjacent to helix H, as opposed to helix D, has the potential to directly influence the initial interaction between protein C inhibitor (PCI) and proteases, as it is located at the top of the serpin.

Slow thrombin in solution.

Abstract: As a tool for understanding biological mechanisms, X-ray crystallography possesses unparalleled power to enlighten, resolve controversy and shift a field of study on to a secure new paradigm. Thanks largely to developments in crystallographic methods, the technique has become accessible to the general biochemist and we have thus witnessed an exponential increase in the number of protein structures deposited every year. It is now commonplace for several structures to be published of the same protein under different crystallization conditions, sometimes resulting in conflicting mechanistic interpretations. Such a controversy has arisen over thrombin's conformational response to Na+ co-ordination, and in this issue of Biochemical Journal, De Filippis and colleagues put the two structural models of thrombin allostery to the test by returning to the techniques of solution biochemistry.