Allosteric Activation of Coagulation Factor VIIa Visualized by Hydrogen Exchange

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, TFbound 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 50 Å 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. Coagulation factor VII (FVII)2 circulates in the blood with ϳ1% in a two-chain form (FVIIa) and the remainder as singlechain zymogen FVII. FVIIa consists of a trypsin-like protease domain and an N-terminal light chain composed of a membrane-binding ␥-carboxyglutamate-rich domain (Gla domain) and two epidermal growth factor-like domains (EGF1 and EGF2) (1). Upon tissue injury, tissue factor (TF) becomes exposed, and the TF⅐FVIIa complex forms and serves as the initiator of the blood coagulation cascade (2). Numerous coagulation proteases function optimally only when complexed to cofactors, and substantial biochemical evidence supports the concept that several of these cofactors induce allosteric changes 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. Similar to the trypsinogen-trypsin pair, zymogen FVII is converted to FVIIa by proteolysis of an internal peptide bond. A canonical "activation domain" was defined in trypsin, which includes the N terminus created upon endoproteolytic activation and three loops referred to as the activation loops (3). In trypsin, the newly generated N-terminal tail spontaneously inserts itself into a cavity close to the three activation loops, termed the activation pocket, resulting in the formation of a critical salt bridge between the N-terminal Ile-16 and Asp-194 of the active site or and in FVIIa (the chymotrypsin numbering is denoted in superscript with parentheses). This salt bridge leads to the formation of a correctly assembled S1 pocket of the active site and full activity. FVIIa, however, has very low activity following endoproteolytic activation and does not spontaneously rearrange into the active form. The three-dimensional structure of FVIIa bound to TF has been solved providing important structural details of the complex and the active form of FVIIa (4). In the complex, parts of the light chain a...

The Origins of Enhanced Activity in Factor VIIa Analogs and the Interplay between Key Allosteric Sites Revealed by Hydrogen Exchange Mass Spectrometry

“…The conformations of zymogen FVII and FVIIa were highly similar and confirmed the zymogenicity of FVIIa, whereas TF binding induced widespread conformational changes in multiple regions of the protease domain, e.g. the N terminus, the activation pocket, and the activation loops (10).…”

“…Although the HX of the light chain of the FVIIa analogs was largely identical to that of FVIIa (data not shown), 4 the protection from HX could be localized to numerous functionally important segments of the FVIIa protease domain, many of which were previously found to be exchange-protected during the allosteric response induced by TF binding (10) (Fig. 3).…”

“…Sample preparations of FVIIa variants and soluble human TF-(1-219) (sTF) were done as described previously (10). Amide 1 H/ 2 H HX was performed exactly as described earlier (10) and initiated by a 12-fold dilution of a protiated protein stock solution in the presence or absence of cofactor into the corresponding deuterated buffer (i.e.…”

“…Quenched samples were immediately frozen in liquid N 2 and stored at Ϫ80°C. The apparatus used for rapid desalting and subsequent mass analysis was configured as described (10). Positive ion-electrospray ionization mass spectra of eluted peptides were acquired on a LCT mass spectrometer (Waters Corp.).…”

“…1) (7)(8)(9). Recently, insights into zymogen FVII and the zymogen-like form of FVIIa were obtained by probing the solution structures of these two activation states, and that of TF-bound FVIIa, by hydrogen exchange monitored by mass spectrometry (HX-MS) (10). The conformations of zymogen FVII and FVIIa were highly similar and confirmed the zymogenicity of FVIIa, whereas TF binding induced widespread conformational changes in multiple regions of the protease domain, e.g.…”