February 26, 2026: Vladimir Kolyadko,The Children’s Hospital of Philadelphia

Описание: Cryo-EM of Liposome-Assembled Macromolecular Complexes to Reveal Protein-Membrane Interactions in Hemostasis

Abstract: Biological membranes containing phosphatidylserine provide a template for the assembly of macromolecular complexes involved in cell signaling and extracellular enzymatic cascades. Our study focuses on the enzymatic complex of the intrinsic Xase (‘ten-ase’), which regulates blood clot formation by assembling on cellular membranes via the high-affinity binding of a cofactor protein, coagulation factor VIIIa. The membrane-bound cofactor engages its cognate serine proteinase, coagulation factor IXa, and accelerates the proteolytic activation of coagulation factor X by a million-fold. The requirement for a membrane surface poses an obstacle to structural studies of the molecular mechanisms of specificity and allostery in the coagulation system. To overcome this, we use small unilamellar vesicles (liposomes) to assemble purified recombinant coagulation factors into a complex for single-particle cryo-EM reconstruction. Conceptually, the use of liposomes allows the cryo-EM structure of the intrinsic Xase to be interpreted in the context of functional studies conducted with the same vesicles. Moreover, the liposomes provide high contrast for picking liposome-decorating protein particles, potentially protect the particles from the air-water interface, and may improve the angular distribution of views of the protein on a sphere. By using this approach, we determined sub-3Å structures of coagulation cofactors in a membrane-bound form and in complex with the proteinase. These structures reveal the mode of protein docking on the membrane and highlight the sites of specific interactions with phosphatidylserine.