Abstract
Membranes containing phosphatidylserine are crucial for the assembly of the intrinsic Xase complex of factors VIIIa and IXa at the site of vascular injury. Conflicting models have been proposed for membrane binding and the molecular architecture of the complex. Molecular mechanisms underlying how the components of intrinsic Xase interact and function have remained elusive due to the challenges posed by membranes in structural studies. Here, we utilize liposomes containing phosphatidylcholine and phosphatidylserine to determine the molecular mechanisms of factor VIII (FVIII) assembly into a membrane-bound complex by using cryo-EM. The structure of FVIII in the absence of membranes (apo-FVIII) reveals the expected organization of the A1, A2, and A3 domains in a pseudo-symmetrical rosette positioned on a base formed by the C1 and C2 domains. The cryo-EM map of apo-FVIII indicates variable motility in the C1 and C2 domains. In the structure of membrane-bound FVIII, its interaction with the membrane is limited to the C1 and C2 domains, which are now stabilized in a single conformation. There are direct contacts between the membrane surface and Lys2092, Phe2093, Met2199, Phe2200, Arg2215, Leu2251, and Leu2252. These residues occupy the level of phospholipid headgroups, remain parallel to the membrane surface, and, contrary to previous proposals, do not insert into the hydrophobic bilayer. Surprisingly, while C1 and C2 are stabilized, membrane binding increases the motility of the A2 domain. In the structure of FVIII bound to factor IXa (FIXa) on the membrane surface, the essential features of the FVIII-membrane interaction are unchanged, although the A2 domain is further stabilized by FIXa. This structure reveals an elongated conformation of FIXa, where its protease domain is lifted above the membrane surface by a stalk formed by the Gla, EGF1, and EGF2 domains and their extensive interactions with FVIII. The structure of the complex demonstrates that the Gla-domain of FIXa interacts with the membrane and with the C1 domain of FVIII; the EGF1 domain interacts with the groove between FVIII C1 and FVIII A3 domains; the EGF2 domain contacts the FVIII A3 domain; the serine proteinase domain interacts via its exosite with the groove between the A2 and A3 domains of FVIII. No direct contacts between FIXa and the A1 or C2 domains of FVIII are evident in the resolved structure. Comparison to the published structure of the FVIII-VWF complex reveals that the Gla and EGF1 domains of FIXa bind to the site occupied by the VWF D' domain, while the membrane-binding sites in FVIII overlap with the site occupied by the VWF D3 domain. This explains why VWF blocks membrane binding by FVIII in circulation. Previous proposals of how FVIII interacts with membranes are now challenged by this series of cryo-EM structures determined at the atomic resolution. Our work also reveals the extended interface between FVIII and FIXa that accounts for their high-affinity interaction on the membrane surface. The molecular architecture revealed by the resolved structures provides atomic details of factor VIII binding to membranes and sheds light on the assembly of the intrinsic Xase.
