CONFORMATIONAL STABILITY OF FACTOR VIIA - BIOPHYSICAL STUDIES OF THERMAL AND GUANIDINE HYDROCHLORIDE-INDUCED DENATURATION

The binding of the multidomain protein factor VIIa (fVIIa) to tissue f actor provides the interprotein communication necessary to make fIIa a n efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual do mains in fIIa and the influence of Ca2+ and an irreversible active-sit e inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochlo ride (GuHCl)-induced unfolding monitored by tryptophan fluorescence an d far-UV circular dichroism (CD) demonstrated that the gamma-carboxygl utamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine proteas e (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the for mation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is sta bilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR -chloromethyl ketone). Thermal unfolding monitored by differential sca nning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain sli ghtly, increasing the unfolding temperature by 2.7 degrees C. In addit ion, Ca2+ is required for a large enthalpy change concomitant with unf olding of the Gla domain, and this unfolding enthalpy is only detectab le in the presence of the SP domain, indicating some kind of interacti on between these domains. Thermal unfolding measured by CD indicates s econdary structural changes at the same temperature as the heat absorp tion in the DSC but only when both the Gla domain and the SP domain ar e present together with Ca2+ ions. Taken together, these results indic ate a Ca2+-dependent interaction between the Gla domain and the SP dom ain, implying a high degree of flexibility of the domains in free fVII a. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreove r, already at physiological temperature, subtle structural changes tak e place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.