Conformational Dynamics of Antithrombin III With its Allosteric Activator Heparin

Abstract

Antithrombin III (ATIII) is a serpin that is involved in the regulation of blood coagulation through the inhibition of blood clotting enzymes. Heparin is an allosteric activator of ATIII that binds to helix-D and causes a conformational change in the reactive center loop (RCL), expelling it from its position partially inserted into beta-sheet A. RCL expulsion in turn increases ATIII activity toward fXa several hundred-fold. Hydrogen/deuterium exchange and mass spectrometry were used to probe the dynamics of ATIII in the presence and absence of a synthetic heparin pentasaccharide (Fondaparinux). Results of our initial hydrogen/deuterium exchange mass spectrometry experiments provide direct, solution phase evidence that heparin cofactor binding alters conformational dynamics in four specific regions of the antithrombin molecule. (1) Helix D -- Heparin binding reduced H/D, consistent with hD extension upon cofactor binding. (2) Breach region -- Beta strands 3A and 5A, flanking the site for RCL insertion into sheet A, showed reduced H/D exchange, consistent with increased rigidity of the breach region and stabilization of a loop-expelled form. (3) Proximal RCL and hinge region -- H/D exchange for residues 376-387, which includes the N-terminal hinge region of the RCL, increased in the presence of heparin, indicating greater solvent exposure and expulsion from beta sheet A. (4) Distal RCL -- Deuterium exchange for 388-402, which includes s1C and the distal side of the RCL, decreased significantly in the presence of heparin, suggesting that s1C extension-mediated stabilization on the C-terminal side of RCL contributes to exposure of the proximal its end upon cofactor binding. Thus, dynamic H/D exchange studies of free and heparin-bound antithrombin molecules in solution will be useful for validating and refining models of ATIII heparin activation inferred from crystal structures.