Loop dynamics of the extracellular domain of human tissue factor and activation of factor VIIa.
In the crystal structure of the complex between the soluble extracellular domain of tissue factor (sTF) and active-site-inhibited VIIa, residues 91 and 92 in the Pro(79)-Pro(92) loop of sTF interact with the catalytic domain of VIIa. It is not known, however, whether this loop has a role in allosteric activation of VIIa. Time-resolved fluorescence anisotropy measurements of probes covalently bound to sTF mutants E84C and T121C show that binding uninhibited Factor VIIa affects segmental motions in sTF. Glu(84) resides in the Pro(79)-Pro(92) loop, and Thr(121) resides in the turn between the first and second antiparallel beta-strands of the sTF subdomain that interacts with the Gla and EGF1 domains of VIIa; neither Glu(84) nor Thr(121) makes direct contact with VIIa. Probes bound to T121C report limited segmental flexibility in free sTF, which is lost after VIIa binding. Probes bound to E84C report substantial segmental flexibility in the Pro(79)-Pro(92) loop in free sTF, which is greatly reduced after VIIa binding. Thus, VIIa binding reduces dynamic motions in sTF. In particular, the decrease in the Pro(79)-Pro(92) loop motions indicates that loop entropy has a role in the thermodynamics of the protein-protein interactions involved in allosteric control of VIIa activation.
Minazzo
Agnese S
AS
Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Dynamics, The University of Montana, Missoula, Montana, USA.
Darlington
Reuben C
RC
Ross
J B Alexander
JB
D015942:Factor VIIa / Q000737:chemistry* / Q000378:metabolism*; D005454:Fluorescence Polarization; D006801:Humans; D008956:Models, Chemical; D015233:Models, Statistical; D009154:Mutation; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D013925:Thromboplastin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D009282:Naphthalenesulfonates; C006421:1,5-I-AEDANS; D013925:Thromboplastin; D015942:Factor VIIa