1 Department of Pharmacy, Faculty of Pharmaceutical Sciences, Københavns Universitet2 Department of Clinical Medicine, Faculty of Health and Medical Sciences, Københavns Universitet3 Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet4 Aalborg University5 Forskningsenheden for Almen Praksis, Eksterne centre, Københavns Universitet6 unknown7 Aalborg Universitetsbibliotek8 Institut for Informations- og Medievidenskab, Aarhus Universitet9 Analytical Biosciences, Department of Pharmacy, Faculty of Health and Medical Sciences, Københavns Universitet10 Aalborg University11 Forskningsenheden for Almen Praksis, Eksterne centre, Københavns Universitet
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.
Journal of Biological Chemistry, 2006, Vol 281, Issue 32, p. 23018-24