title: NMR resonance assignments of thrombin reveal the conformational and dynamic effects of ligation.
abstract: The serine protease thrombin is generated from its zymogen prothrombin at the end of the coagulation cascade. Thrombin functions as the effector enzyme of blood clotting by cleaving several procoagulant targets, but also plays a key role in attenuating the hemostatic response by activating protein C. These activities all depend on the engagement of exosites on thrombin, either through direct interaction with a substrate, as with fibrinogen, or by binding to cofactors such as thrombomodulin. How thrombin specificity is controlled is of central importance to understanding normal hemostasis and how dysregulation causes bleeding or thrombosis. The binding of ligands to thrombin via exosite I and the coordination of Na(+) have been associated with changes in thrombin conformation and activity. This phenomenon has become known as thrombin allostery, although direct evidence of conformational change, identification of the regions involved, and the functional consequences remain unclear. Here we investigate the conformational and dynamic effects of thrombin ligation at the active site, exosite I and the Na(+)-binding site in solution, using modern multidimensional NMR techniques. We obtained full resonance assignments for thrombin in seven differently liganded states, including fully unliganded apo thrombin, and have created a detailed map of residues that change environment, conformation, or dynamic state in response to each relevant single or multiple ligation event. These studies reveal that apo thrombin exists in a highly dynamic zymogen-like state, and relies on ligation to achieve a fully active conformation. Conformational plasticity confers upon thrombin the ability to be at once selective and promiscuous.
authors.affiliation: Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 0XY, United Kingdom.
authors.forename: Bernhard C
authors.initials: BC
authors.lastname: Lechtenberg
authors.forename: Daniel J D
authors.initials: DJ
authors.lastname: Johnson
authors.forename: Stefan M V
authors.initials: SM
authors.lastname: Freund
authors.forename: James A
authors.initials: JA
authors.lastname: Huntington
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D004792:Enzyme Precursors / Q000737:chemistry; D006801:Humans; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D056004:Molecular Dynamics Simulation; D011485:Protein Binding; D011487:Protein Conformation; D013379:Substrate Specificity; D013917:Thrombin / Q000737:chemistry*
chemical_list: D004792:Enzyme Precursors; D008024:Ligands; D013917:Thrombin