A combined structural dynamics approach identifies a putative switch in factor VIIa employed by tissue factor to initiate blood coagulation.
Coagulation factor VIIa (FVIIa) requires tissue factor (TF) to attain full catalytic competency and to initiate blood coagulation. In this study, the mechanism by which TF allosterically activates FVIIa is investigated by a structural dynamics approach that combines molecular dynamics (MD) simulations and hydrogen/deuterium exchange (HX) mass spectrometry on free and TF-bound FVIIa. The differences in conformational dynamics from MD simulations are shown to be confined to regions of FVIIa observed to undergo structural stabilization as judged by HX experiments, especially implicating activation loop 3 (residues 365-374{216-225}) of the so-called activation domain and the 170-loop (residues 313-322{170A-175}) succeeding the TF-binding helix. The latter finding is corroborated by experiments demonstrating rapid deglycosylation of Asn322 in free FVIIa by PNGase F but almost complete protection in the presence of TF or an active-site inhibitor. Based on MD simulations, a key switch of the TF-induced structural changes is identified as the interacting pair Leu305{163} and Phe374{225} in FVIIa, whose mutual conformations are guided by the presence of TF and observed to be closely linked to the structural stability of activation loop 3. Altogether, our findings strongly support an allosteric activation mechanism initiated by the stabilization of the Leu305{163}/Phe374{225} pair, which, in turn, stabilizes activation loop 3 and the S(1) and S(3) substrate pockets, the activation pocket, and N-terminal insertion.
Olsen
Ole H
OH
Department of Haemostasis Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark. oho@novonordisk.com
Rand
Kasper D
KD
Østergaard
Henrik
H
Persson
Egon
E
D001777:Blood Coagulation / Q000502:physiology*; D015942:Factor VIIa / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D013058:Mass Spectrometry; D008958:Models, Molecular; D011487:Protein Conformation; D013925:Thromboplastin / Q000737:chemistry*
D013925:Thromboplastin; D015942:Factor VIIa