Abstract
Coagulation factor VIIa (FVIIa) is a serine protease that, after binding to tissue factor (TF), plays a pivotal role in the initiation of blood coagulation. We used hydrogen exchange monitored by mass spectrometry to visualize the details of FVIIa activation by comparing the exchange kinetics of distinct molecular states, namely zymogen FVII, endoproteolytically cleaved FVIIa, TF-bound zymogen FVII, TF-bound FVIIa, and FVIIa in complex with an active site inhibitor. The hydrogen exchange kinetics of zymogen FVII and FVIIa are identical indicating highly similar solution structures. However, upon tissue factor binding, FVIIa undergoes dramatic structural stabilization as indicated by decreased exchange rates localized throughout the protease domain and in distant parts of the light chain, spanning across 50A and revealing a concerted interplay between functional sites in FVIIa. The results provide novel insights into the cofactor-induced activation of this important protease and reveal the potential for allosteric regulation in the trypsin family of proteases.
Highlights
In the conformation of their cognate enzymes (1, 2)
hydrogen exchange (HX) experiments were initiated by dilution of proteins into deuterium, and the subsequent time-resolved deuterium incorporation was localized to various regions of FVII/factor VIIa (FVIIa) by mass analysis of peptides produced by peptic proteolysis
To ensure a correlation between the experimental conditions and those found in the blood, the HX kinetics of FVIIa and tissue factor (TF)-bound FVIIa were monitored at physiologi
Summary
In the conformation of their cognate enzymes (1, 2). TF functions to localize FVIIa and as an allosteric regulator, which dramatically enhances the activity of FVIIa. We studied the HX kinetics of FVIIa in the presence of an active site inhibitor (IN18), which is derivatized from the amidinophenyl family of inhibitors (11) known to lock FVIIa in the active conformation (5). Both TF and IN18 induce the active conformation but bind at different sites on FVIIa, and the latter data set is. Regions that are affected by both molecules represent regions in FVIIa responding to activation, whereas regions that respond to only one of the molecules can be disregarded as due to the steric effects at binding interfaces With these tools, we were able to characterize the structural properties of the different states of FVII/FVIIa, which are of biological relevance during activation in the blood. Regions of FVIIa undergoing structural rearrangements or changes in dynamics upon transition to the active form are revealed, providing novel insights into the molecular nature of the allosteric activation of this cofactor-dependent trypsin-like protease
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