Importance of factor VIIa Gla-domain residue Arg-36 for recognition of the macromolecular substrate factor X Gla-domain

Macromolecular substrate docking with coagulation enzyme-cofactor complexes involves multiple contacts distant from the enzyme's catalytic cleft. Here we characterize the binding of the Gla-domain of macromolecular substrate coagulation factor X to the complex of tissue factor (TF) and VIIa. Site-directed mutagenesis of charged residue side chains in the VIIa Cia-domain identified Arg-36 as being important for macromolecular substrate docking. Ala substitution for Arg-36 resulted in an increased K-M and a decreased rate of X activation. X with a truncated Gla-domain was activated by mutant and wild-type VIIa at indistinguishable rates, demonstrating that Arg-36 interactions require a properly folded Cia-domain of the macromolecular substrate. VIIa Arg-36 was also required for effective docking of the X Cia-domain in the absence of phospholipid, demonstrating that the Cia-domain of VIIa participates in protein-protein interactions with X. In the absence of TF, the mutant VIIa had essentially normal function, indicating that the cofactor positions VIIa's Gla-domain for optimal macromolecular substrate docking. Computational docking suggests multiple charge complementary contacts of the X Cia-domain with TF-VIIa. A prominent interaction is made by the functionally important X residue Gla-14 with the center of the extended docking site created by residues in the carboxyl module of TF and the contiguous VIIa Cia-domain. These data demonstrate the functional importance of interactions of the Cia-domains of enzyme and substrate, and begin to elucidate the molecular details of the ternary TF VIIa X complex.