Regulation of the Catalytic Function of Coagulation Factor VIIa by a Conformational Linkage of Surface Residue Glu 154 to the Active Site ² Justin Shobe, Craig D. Dickinson, and Wolfram Ruf* Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla, California 92037 ReceiVed August 12, 1998; ReVised Manuscript ReceiVed December 16, 1998 ABSTRACT: 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. The serine protease family is essential for a variety of physiological and pathological processes in higher organisms. These enzymes are typically synthesized as zymogen precur- sors that are activated by proteolytic cleavage of a specific scissile bond, resulting in conformational changes that lead to catalytic activation. The zymogen structures are character- ized by several segments, designated the activation domain, that display increased flexibility compared to their enzyme structure counterparts (1). The restriction of conformational flexibility, in particular, the ion pair formation of the R-amino group of Ile 16 with Asp 194 (residue numbering is based on chymotrypsin positions), is essential for full catalytic activity of a serine protease domain. The coagulation serine proteases are a subclass of trypsin-like serine proteases with highly restricted substrate specificity. In addition, the pro- teolytic function of these enzymes frequently depends on protein cofactors that support macromolecular substrate binding and allosterically influence substrate specificity. Factor VIIa (VIIa) 1 serves as the initiating protease of the coagulation cascade by binding to the cell surface receptor and catalytic cofactor tissue factor (TF), a member of the cytokine receptor family. The structure of the TF‚VIIa complex (2) and extensive site-directed mutagenesis of TF and VIIa (3, 4) have defined the precise molecular interac- tions between cofactor and enzyme. The integration of these structure-function studies suggests that macromolecular substrate factor X must assemble through multiple contacts with the complex (5). The creation of an extended macro- molecular substrate docking site by regions of cofactor and enzyme, in part, explains the low proteolytic activity of VIIa in the absence of cofactor. Furthermore, VIIa’s catalytic activity toward small peptide substrates is low, but dramati- cally enhanced by binding of TF (6-8), indicating a direct allosteric regulation of the active site by cofactor interactions. Because the amino-terminal Ile 16 is susceptible to chemical modification in free VIIa, but not in the TF-bound enzyme (9), a zymogen-like conformation of VIIa in the absence of cofactor is the likely reason for the low amidolytic activity. The cofactor-dependent allosteric activation of the VIIa protease domain is mediated by specific contacts in the interface with TF, in particular Met 164 (2, 4). The localization of this interface adjacent to regions of the activation domain (the segment of residues 182-189) has led to the proposal that cofactor interactions enhance the catalytic function of VIIa by supporting the labile zymogen transition to the active enzyme (4, 10). Inhibitor binding to the catalytic cleft of VIIa also decreases the susceptibility of Ile 16 to chemical modifica- tions (10), indicating that cofactor interactions and active site occupancy induce a similar conformational transition in the protease domain of VIIa. Conformational changes associated with formation of a stabilized amino-terminal Ile 16 ion pair with Asp 194 likely affect the local environment around the insertion site. The amino-terminal insertion site is part of the epitope of a conformation-sensitive monoclonal ² Supported by NIH Grants R01 HL48752 and P01 HL16411. Performed during the tenure of an Established Investigator Award from the American Heart Association (to W.R.). * Corresponding author: Departments of Immunology and Vascular Biology, IMM-17, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Telephone: (619) 784-2748. Fax: (619) 784-8480. E-mail: ruf@scripps.edu. 1 Abbreviations: TF, tissue factor; VII and VIIa, coagulation factor VII and VIIa, respectively; FFR-VIIa, VIIa modified with the active site inhibitor Phe-Phe-Arg chloromethyl ketone; PC, phosphatidylcho- line vesicles; PCPS, phosphatidylcholine/phosphatidylserine vesicles. 2745 Biochemistry 1999, 38, 2745-2751 10.1021/bi981951g CCC: $18.00 © 1999 American Chemical Society Published on Web 02/10/1999