Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II.

Verhamme IM, Olson ST, Tollefsen DM, Bock PE
J Biol Chem. 2002 277 (9): 6788-98

PMID: 11724802 · DOI:10.1074/jbc.M110257200

The substrate specificity of thrombin is regulated by binding of macromolecular substrates and effectors to exosites I and II. Exosites I and II have been reported to be extremely linked allosterically, such that binding of a ligand to one exosite results in near-total loss of affinity for ligands at the alternative exosite, whereas other studies support the independence of the interactions. An array of fluorescent thrombin derivatives and fluorescein-labeled hirudin(54-65) ([5F]Hir(54-65)(SO(3)(-))) were used as probes in quantitative equilibrium binding studies to resolve whether the affinities of the exosite I-specific ligands, Hir(54-65)(SO(3)(-)) and fibrinogen, and of the exosite II-specific ligands, prothrombin fragment 2 and a monoclonal antibody, were affected by alternate exosite occupation. Hir(54-65)(SO(3)(-)) and fibrinogen bound to exosite I with dissociation constants of 16-28 nm and 5-7 microm, respectively, which were changed < or =2-fold by fragment 2 binding. Native thrombin and four thrombin derivatives labeled with different probes bound fragment 2 and the antibody with dissociation constants of 3-12 microm and 1.8 nm, respectively, unaffected by Hir(54-65)(SO(3)(-)). The results support a ternary complex binding model in which exosites I and II can be occupied simultaneously. The thrombin catalytic site senses individual and simultaneous binding of exosite I and II ligands differently, resulting in unique active site environments for each thrombin complex. The results indicate significant, ligand-specific allosteric coupling between thrombin exosites I and II and catalytic site perturbations but insignificant inter-exosite thermodynamic linkage.

MeSH Terms (20)

Allosteric Site Antibodies, Monoclonal Binding, Competitive Binding Sites Catalytic Domain Dose-Response Relationship, Drug Fibrinogen Hirudins Humans Kinetics Ligands Models, Chemical Peptides Protein Binding Protein Conformation Spectrometry, Fluorescence Substrate Specificity Thermodynamics Thrombin Time Factors

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