Conformation of factor Xa in solution revealed by single molecule spectroscopy

Abstract

All current X-ray structures of fXa are devoid of the γ-carboxyglutamate (Gla) domain and fail to reveal the overall conformation of the free protein. The recent cryo-EM structure of fXa in the prothrombinase complex is the only structure of full length fXa and shows that the Gla domain is positioned at an angle relative to the EGF1 domain. Establish if the curved conformation of fXa revealed by cryo-EM is also present in solution. The conformation of fXa in solution is studied by single molecule Förster resonance energy transfer (smFRET). The conformation of full length fXa in solution is resolved for the first time. The conformation is curved and extremely sensitive to Ca2+. It does not differ significantly from its zymogen form or from that present in the prothrombinase complex free or bound to the physiological substrates prothrombin and meizothrombin. Measurements by smFRET reveal that fXa has a curved conformation in solution, free or bound to physiological ligands, and validate the recent cryo-EM structures of prothrombinase. The drastic conformational changes observed in the absence of Ca2+ suggest that the structural architecture of fXa changes upon administration of vitamin-K antagonists that perturb the interaction of the Gla domain with divalent cations.