title: Binding of exosite ligands to human thrombin. Re-evaluation of allosteric linkage between thrombin exosites I and II.
abstract: 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.
authors.affiliation: Department of Pathology, Vanderbilt University School of Medicine, Medical Center North, Nashville, TN 37232, USA.
authors.forename: Ingrid M
authors.initials: IM
authors.lastname: Verhamme
authors.forename: Steven T
authors.initials: ST
authors.lastname: Olson
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Tollefsen
authors.forename: Paul E
authors.initials: PE
authors.lastname: Bock
mesh_terms: D000495:Allosteric Site; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D020134:Catalytic Domain; D004305:Dose-Response Relationship, Drug; D005340:Fibrinogen / Q000737:chemistry; D006629:Hirudins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D013816:Thermodynamics; D013917:Thrombin / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000911:Antibodies, Monoclonal; D006629:Hirudins; D008024:Ligands; D010455:Peptides; D005340:Fibrinogen; D013917:Thrombin