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

Abstract

The protease domain of coagulation factor VIIa (FVIIa) is homologous to trypsin with a similar active site architecture. The catalytic function of FVIIa is regulated by allosteric modulations induced by binding of divalent metal ions and the cofactor tissue factor (TF). To further elucidate the mechanisms behind these transformations, the effects of Zn²⁺ binding to FVIIa in the free form and in complex with TF were investigated. Equilibrium dialysis suggested that two Zn²⁺ bind with high affinity to FVIIa outside the N-terminal γ-carboxyglutamic acid (Gla) domain. Binding of Zn²⁺ to FVIIa, which was influenced by the presence of Ca²⁺, resulted in decreased amidolytic activity and slightly reduced affinity for TF. After binding to TF, FVIIa was less susceptible to zinc inhibition. Alanine substitutions for either 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⁺ binding sites in the Glu210–Glu220 Ca²⁺ binding loop adjacent to the so-called activation domain canonical to serine proteases. Based on our results, we propose a model that describes the conformational changes underlying the Zn²⁺-mediated allosteric down-regulation of FVIIa's activity.