SummaryAntithrombin (AT) is the most important inhibitor of the coagulation proteases. Its activity is stimulated by glycosaminoglycans such as heparin, through allosteric and template mechanisms. AT utilises an induced-fit mechanism to bind with high affinity to a pentasaccharide sequence found in about one-third of heparin chains. The conformational changes behind this mechanism have been characterised by several crystal structures of AT in the absence and presence of the pentasaccharide. Pentasaccharide binding ultimately results in a conformational change that improves affinity by about 1000-fold. Crystal structures show several differences, including the expulsion of the hinge region of the reactive centre loop from β-sheet A, known to be critical for the allosteric activation of AT. Here we present data that reveals an energetically distinct intermediate on the path to full activation, where the majority of conformational changes have already occurred. A crystal structure of this intermediate shows that the hinge region is in a native like state, in spite of having the pentasaccharide bound in the normal fashion. We engineered a disulphide bond to lock the hinge in its native position to determine the energetic contributions of the initial and final conformational events. Approximately 60% of the free energy contribution of conformational change is provided by the final step of hinge region expulsion and subsequent closure of the main β-sheet A. A new analysis of the individual structural changes provides a plausible mechanism for propagation of conformational change from the heparin binding site to the remote hinge region in β-sheet A.
Antithrombin requires allosteric activation by heparin for efficient inhibition of its target protease, factor Xa. A pentasaccharide sequence found in heparin activates antithrombin by inducing conformational changes that affect the reactive center of the inhibitor resulting in optimal recognition by factor Xa. The mechanism of transmission of the activating conformational change from the heparin-binding region to the reactive center loop remains unresolved. To investigate the role of helix D elongation in the allosteric activation of antithrombin, we substituted a proline residue for Lys(133). Heparin binding affinity was reduced by 25-fold for the proline variant compared with the control, and a significant decrease in the associated intrinsic fluorescence enhancement was also observed. Rapid kinetic studies revealed that the main reason for the reduced affinity for heparin was an increase in the rate of the reverse conformational change step. The pentasaccharide-accelerated rate of factor Xa inhibition for the proline variant was 10-fold lower than control, demonstrating that the proline variant cannot be fully activated toward factor Xa. We conclude that helix D elongation is critical for the full conversion of antithrombin to its high affinity, activated state, and we propose a mechanism to explain how helix D elongation is coupled to allosteric activation.
Antithrombin is unique among the serpins in that it circulates in a native conformation that is kinetically inactive toward its target proteinase, factor Xa. Activation occurs upon binding of a specific pentasaccharide sequence found in heparin that results in a rearrangement of the reactive center loop removing constraints on the active center P1 residue. We determined the crystal structure of an activated antithrombin variant,
Antithrombin requires heparin for efficient inhibition of the final two proteinases of the blood coagulation cascade, factor Xa and thrombin. Antithrombin binds heparin via a specific pentasaccharide domain in a twostep mechanism whereby initial weak binding is followed by a conformational change and subsequent tight binding. The goal of this study is to investigate the role of a reducing-end extension in the binding of the longer oligosaccharides that contain the cognate pentasaccharide sequence. We determined the antithrombin binding properties of a synthetic heptasaccharide containing the natural pentasaccharide sequence (DEFGH) and an additional reducing-end disaccharide (DEFGHGH). Binding at low ionic strength is unaffected by the disaccharide addition, but at ionic strengths >0.2 the mode of heptasaccharide binding changes resulting in a 2-fold increase in affinity due to a decrease in the off-rate caused by a greater nonionic contribution to binding. Molecular modeling of possible binding modes for the heptasaccharide at high ionic strength indicates a possible shift in position of the pentasaccharide domain to occupy the extended heparin-binding site. This conclusion supports the likely presence of a range of sequences that can bind to and activate antithrombin in the natural heparan sulfates that line the vascular endothelium.The anticoagulant effect of the widely prescribed drug heparin is mediated by its binding to and activation of the serpin antithrombin against the final two proteinase in the blood coagulation cascade, factor Xa and thrombin. Specific binding to heparin is due to the presence of a unique pentasaccharide that is found in about a third of the heparin chains contained in commercial heparin preparations. Pentasaccharide binding by antithrombin is a two-step process where, after the rapid formation of an initial, weak complex, antithrombin changes conformation to form a tight complex (1). The conformational change in antithrombin also affects the reactive center loop and releases constraints on the P1 arginine residue that can then be recognized by target proteinases (2), thus resulting in a 300-fold acceleration in the rate of factor Xa inactivation, whereas full-length heparin is required for acceleration of the inactivation of thrombin since thrombin must also bind heparin via its exosite 2 (3, 4). Due to the detrimental side effects of long chain therapeutic heparin preparations, drug design has focused on maximization of the anti-factor Xa activity of heparin and minimization of the anti-thrombin activity.It has been proposed that the mechanism by which pentasaccharide binding affects conformational change in the reactive center loop of antithrombin involves binding to and elongation of the C terminus of helix D (5). The recently solved structure of antithrombin in complex with a high affinity form of the pentasaccharide (6) indicated that the C terminus of helix D was not directly involved in pentasaccharide binding and was more likely involved in the binding of the non-reducing-e...
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