Binding of Zn2+ to a Ca2+ loop allosterically attenuates the activity of factor VIIa and reduces its affinity for tissue factor

The protease domain of coagulation factor VIIa (FVIIa) is homologous to try psin with a similar active site architecture. The catalytic function of FVI Ia is regulated by allosteric modulations induced by binding of divalent me tal ions and the cofactor tissue factor (TF). To further elucidate the mech anisms behind these transformations, the effects of Zn2+ binding to FVIIa i n the free form and in complex with TF were investigated. Equilibrium dialy sis suggested that two Zn2+ bind with high affinity to FVIIa outside the N- terminal gamma-carboxyglutamic acid (Gla) domain. Binding of Zn2+ to FVIIa, which was influenced by the presence of Ca2+, resulted in decreased amidol ytic activity and slightly reduced affinity for TF. After binding to TF, FV IIa was less susceptible to zinc inhibition. Alanine substitutions for eith er of two histidine residues unique for FVIIa, His216, and His257, produced FVIIa variants with decreased sensitivity to Zn2+ inhibition. A search for putative Zn2+ binding sites in the crystal structure of the FVIIa protease domain was performed by Grid calculations. We identified a pair of Zn2+ bi nding sites in the Glu210-Glu220 Ca2+ binding loop adjacent to the so-calle d activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Z n2+-mediated allosteric down-regulation of FVIIa's activity.