Antithrombin III residue tyrosine-131 serves as a molecular switch during heparinmediated conformational activation of its anticoagulant activity

The foundation of this project is the hypothesis that binding of antithrombin III (ATIII) to heparin pentasaccharide (H5) leads to global changes in ATIII structure that starts at its heparin binding site (HBS), propagates throughout the molecule, and culminates with the expulsion of the reactive center loop (RCL) - leading to complete activation of ATIII against Factor Xa (fXa). Structure-based analyses of three ATIII structures led to the identification of residues that are hypothesized to be involved in conformational change propagation. These residues were mutated and analyzed to ascertain their importance. The first series of mutants, Tyr131 ?Ala, Leu, or Phe, were made to determine the effect of helix D elongation-mediated Tyr on conformational change propagation. Secondly, Leu140, which lies directly between the HBS and the RCL, was mutated to a Val or Phe to assess the contribution of this (?-sheet 2A residue in allosteric activation. The third series, comprised of Tyr131?Leu ATIII variants with RCL-deletion/s Val400, Arg399Val400, or Arg399Val400Thr401, look into the potential cooperativity of Y131 and RCL residues in depressing endogenous ATIII anti-fXa activity. The contributions of these residues in ATIII structure and function were evaluated by measuring their thrombin and fXa inhibition rates, determining their native to noninhibitory conformation melting temperature, and assessing their heparin binding affinity. These studies clearly indicate that ATIII allosteric activation is dependent on the investigated residues. Tyr131 and the mutated RCL residues play a crucial role, and act cooperatively, in depressing ATIII anti-fXa activity only. In addition, they are also important in ATIII heparin binding affinity and in maintaining ATIII stability. Additionally, changing Leu140?Phe slightly increased cofactor-independent ATIII fXa inhibition rates, indicating that spacing between helix D and strand 2A promote further conformational adjustments in the molecule, including structural changes that might increase RCL accessibility and/or lead to greater exposure of fXa recognition sites. The mutagenesis studies that were undertaken offer tremendous insight on the conformational events that occur within ATIII following H5 binding and will aid in the development of better anticoagulant therapeutics.