Freskgârd P O, Petersen L C, Gabriel D A, Li X, Persson E
Vessel Wall Biology, Health Care Discovery, Novo Nordisk A/S, Gentofte, Denmark.
Biochemistry. 1998 May 19;37(20):7203-12. doi: 10.1021/bi972847m.
The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized 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 scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.
多结构域蛋白因子VIIa（fVIIa）与组织因子的结合提供了蛋白间通讯，使fVIIa成为血液凝固外源性途径初始事件的高效催化剂。我们研究了fVIIa中各个结构域的稳定性以及Ca2+和不可逆活性位点抑制剂（FFR-氯甲基酮）的影响。通过色氨酸荧光和远紫外圆二色性（CD）监测的平衡盐酸胍（GuHCl）诱导的去折叠表明，γ-羧基谷氨酸（Gla）结构域在0.3 M GuHCl时去折叠，丝氨酸蛋白酶（SP）结构域在3 M GuHCl时去折叠，并且Ca2+是Gla结构域中形成有序紧密结构的先决条件。酰胺水解活性的丧失与第一个转变同时发生，该转变由活性位点抑制剂稳定，并且使用荧光抑制剂（DEGR-氯甲基酮）证明了活性位点环境的变化。通过差示扫描量热法（DSC）监测的热去折叠表明，Ca2+略微稳定了SP结构域，使去折叠温度升高了2.7℃。此外，Ca2+是Gla结构域去折叠时伴随的大焓变所必需的，并且这种去折叠焓仅在存在SP结构域时才可检测到，表明这些结构域之间存在某种相互作用。通过CD测量的热去折叠表明，在与DSC中的吸热相同的温度下发生二级结构变化，但仅当Gla结构域和SP结构域与Ca2+离子一起存在时才会发生。综上所述，这些结果表明Gla结构域和SP结构域之间存在Ca2+依赖性相互作用，这意味着游离fVIIa中结构域具有高度的灵活性。还表明表皮生长因子样结构域在升高的温度和高GuHCl浓度下是稳定的。此外，已经在生理温度下，发生了细微的结构变化，这些变化影响了fVIIa的整体形状并对其酶活性有害。
