Disruption of a Tight Cluster Surrounding Tyrosine 131 in the Native Conformation of Antithrombin III Activates It for Factor Xa Inhibition

Abstract

The native conformation of antithrombin III (ATIII) is a poor inhibitor of its coagulation pathway target enzymes because of the partial insertion of its reactive center loop (RCL) in its central A ␤-sheet. This study focused on tyrosine 131, which is located at the helix D-sheet A interface, adjacent to the ATIII pentasaccharide and heparin cofactor-binding sites and some 17 Å away from the RCL insertion. cluster at the helix D-strand 2A interface of native antithrombin contributes significantly to the stability of the ground state conformation, and tyrosine 131 serves as a heparin-responsive molecular switch during the allosteric activation of ATIII anticoagulant activity.To efficiently inhibit factor Xa (fXa), 2 the anticoagulant protein antithrombin III (ATIII) must first bind a pentasaccharide sequence found in pharmaceutical heparins or vascular wall heparan sulfate proteoglycans (HSPGs) and then undergo a protein conformational change that increases reactivity of its reactive center loop (RCL) and other recognition elements with the target enzyme (1-4). Protein conformational changes like the one underlying heparin-dependent ATIII activation result from reductions in the strengths of an initial set of native ground state structural interactions, which are coupled to increases in the strengths of new structural interactions formed during the course of activation. There may be an uncatalyzed equilibrium between different conformations of a protein. For example, the equilibrium between the RCL-inserted and RCLexpelled forms of ATIII is responsible for its slow progressive inhibition of clotting factors and is an example of an uncatalyzed equilibrium. Alternatively, binding of a cofactor ligand may drive the conformational equilibrium of a protein toward a particular state of the molecule, such as AT*H, the heparinbound and activated form of ATIII. For protein conformational changes driven by ligand binding, initial interactions between the ligand and the protein induce structural rearrangements proximal to the binding site, and these changes promote subsequent distal structural adjustments and conformational changes that are successively transmitted through the molecules until a stable complex is achieved.Our long term goal is to develop a thorough understanding of the allosteric, heparin binding-induced conformational change that propagates through ATIII and activates its anticoagulant activity. Intermediate steps in this project will be to identify key structural interactions that stabilize native, transient intermediate, and activated conformations of antithrombin and that are switched off or on during the activation process. This study describes the identification of one such switch, tyrosine 131, that is located at the interface of helix D and central ␤-sheet A.ATIII helix D (1) contains heparin-binding residues Lys 125 (5), Arg 129 (6), Phe 122 (7), Arg 132 , Lys 133 (8), and Lys 136 (9). Helix D of pentasaccharide-bound, RCL-expelled and activated ATIII (AT*H) (Fig. 1b) is 1.5 turns longer at its C-te...

Activation of antithrombin as a factor IXa and Xa inhibitor involves mitigation of repression rather than positive enhancement

“…A further 6‐fold rate enhancement, together with RCL expulsion, occurred when H5 bound, to give ∼3‐fold higher k 2 than wild‐type antithrombin bound to H5. Significantly, an earlier study in which only Y131 was changed to Ala or Leu found a similar effect, with greatly enhanced basal rate of factor Xa inhibition in the absence of heparin (25 to 29‐fold) and a further 8 to 12‐fold enhancement when H5 bound [9]. Since in both cases most of the activation occurred while the RCL hinge was still constrained by β‐sheet A, this raised doubts about the role of RCL hinge expulsion as the means of activation through engagement of a new favorable exosite.…”

Toggle switch residues control allosteric transitions in bacterial adhesins by participating in a concerted repacking of the protein core

“…Alternative conformation-specific orientations of side chains have been reported in other allosteric proteins, e.g. periplasmic (maltose) binding protein MalE [34], rhodopsin [35], integrin [36], DNA-binding receptor NtrC [37], and anti-thrombin III [38]. However, the consequences of the toggle switch-like effect on the conformation and function of allosteric proteins has not been studied thoroughly, nor are they fully appreciated.…”

Polysulfated Trehalose as a Novel Anticoagulant Agent with Dual Mode of Action

“…Arg145 is placed within the heparin binding site of human AT [ 42 ]. Further, in native AT, Tyr131-Asn127-Leu130-Leu140-Ser142 forms a tight cluster at the helix D-strand 2A interface and tight interactions between Tyr131 and neighbouring hD, and s2A stabilizes the native conformation of AT; it has also been hypothesized and tested that disrupting this cluster would activate AT independently of heparin [ 43 ]. Helix D residues 120–124 make multiple van der Waals contacts with residues 161–166 of helix E, where the rotation of helix D on binding to heparin pivots on the side chain of Phe123 and clashes into helix E. Here, the movement of Tyr166 is considered one of the important events in the propagation of conformational change from the heparin binding site to the distant hinge region of the RCL, required for the allosteric activation of AT [ 44 ].…”