Regulation of the catalytic function of coagulation factor VIIa by a conformational linkage of surface residue Glu 154 to the active site.
Coagulation factor VIIa is an allosterically regulated trypsin-like serine protease that initiates the coagulation pathways upon complex formation with its cellular receptor and cofactor tissue factor (TF). The analysis of a conformation-sensitive monoclonal antibody directed to the macromolecular substrate exosite in the VIIa protease domain demonstrated a conformational link from this exosite to the catalytic cleft that is independent of cofactor-induced allosteric changes. In this study, we identify Glu 154 as a critical surface-exposed exosite residue side chain that undergoes conformational changes upon active site inhibitor binding. The Glu 154 side chain is important for hydrolysis of scissile bond mimicking peptidyl p-nitroanilide substrates, and for inhibition of VIIa's amidolytic function upon antibody binding. This exosite residue is not linked to the catalytic cleft residue Lys 192 which plays an important role in thrombin's allosteric coupling to exosite I. Allosteric linkages between VIIa's active site and the cofactor binding site or between the cofactor binding site and the macromolecular substrate exosite were not influenced by mutation of Glu 154. Glu 154 thus only influences the linkage of the macromolecular substrate binding exosite to the catalytic center. These data provide novel evidence that allosteric regulation of VIIa's catalytic function involves discrete and independent conformational linkages and that allosteric transitions in the VIIa protease domain are not globally coupled.
Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA.
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Shobe
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Dickinson
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Ruf
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D000915:Antibody Affinity / Q000235:genetics; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D002384:Catalysis; D015942:Factor VIIa / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D018698:Glutamic Acid / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013925:Thromboplastin / Q000737:chemistry
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D018698:Glutamic Acid; D013925:Thromboplastin; D015942:Factor VIIa