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.