Roles of N-Terminal Region Residues Lys11, Arg13, and Arg24 of Antithrombin in Heparin Recognition and in Promotion and Stabilization of the Heparin-Induced Conformational Change

Publisher Website
Google Scholar
Abstract
The N-terminal region residues, Lys11, Arg13, and Arg24, of the plasma coagulation inhibitor, antithrombin, have been implicated in binding of the anticoagulant polysaccharide, heparin, from the identification of natural mutants with impaired heparin binding or by the X-ray structure of a complex of the inhibitor with a high-affinity heparin pentasaccharide. Mutations of Lys11 or Arg24 to Ala in this work each reduced the affinity for the pentasaccharide ∼40-fold, whereas mutation of Arg13 to Ala led to a decrease of only ∼7-fold. All three substitutions resulted in the loss of one ionic interaction with the pentasaccharide and those of Lys11 or Arg24 also in 3−5-fold losses in affinity of nonionic interactions. Only the mutation of Lys11 affected the initial, weak interaction step of pentasacharide binding, decreasing the affinity of this step ∼2-fold. The mutations of Lys11 and Arg13 moderately, 2−7-fold, altered both rate constants of the second, conformational change step, whereas the substitution of Arg24 appreciably, ∼25-fold, reduced the reverse rate constant of this step. The N-terminal region of antithrombin is thus critical for high-affinity heparin binding, Lys11 and Arg24 being responsible for maintaining appreciable and comparable binding energy, whereas Arg13 is less important. Lys11 is the only one of the three residues that is involved in the initial recognition step, whereas all three residues participate in the conformational change step. Lys11 and Arg13 presumably bind directly to the heparin pentasaccharide by ionic, and in the case of Lys11, also nonionic interactions. However, the role of Arg24 most likely is indirect, to stabilize the heparin-induced P-helix by interacting intramolecularly with Glu113 and Asp117, thereby positioning the crucial Lys114 residue for optimal ionic and nonionic interactions with the pentasaccharide. Together, these findings show that N-terminal residues of antithrombin make markedly different contributions to the energetics and dynamics of binding of the pentasaccharide ligand to the native and activated conformational states of the inhibitor that could not have been predicted from the X-ray structure.

This publication has 17 references indexed in Scilit: