PS-GC Nanodiscs Assembly for Structural Studies of Coagulation Proteins and Their Complexes

Abstract

Nanodiscs (ND) are well known as a lipid membrane platform for structural and functional studies of transmembrane proteins but they can be also helpful for structural studies of membrane associated proteins. The membrane-bound structure of Factor VIII active form (FVIIIa) within the Factor VIIIa - Factor IXa (Tenase) complex is crucial for normal hemostasis and requires binding to the surface of the negatively charged activated platelet membrane. Despite the critical role of FVIII for coagulation process, the knowledge of its membrane-bound organization alone or within the Tenase complex is incomplete which hampers drug discovery for effective regulation of the complex activity and thus design of new pro- and anti-coagulant drugs. In this study we present our work on ND designed for structural studies of membrane-bound FVIII by transmission electron microscopy (TEM) and single-particle analysis (SPA). ND assembled from galactosylceramide (GC) and negatively charged phosphatidylserine (PS) lipid mixtures, and with two different membrane scaffolding proteins forms (MSP1D1 and MSP1E3D1) were characterized by negatively stained TEM showing that their size and homogeneity strongly depended on the lipid composition and lipid to membrane scaffolding protein ratio. Our results show that the ND with highest PS content (80%) and average diameter of 12 nm are the most suitable for structure determination of the membrane-bound FVIII molecules by single particle analysis. Our preliminary 3D reconstruction of the FVIII bound to the PS containing ND demonstrates the suitability of the optimized ND for structural studies by TEM and SPA. Further assembly of the FVIIIa and the whole FVIIIa-FIXa complex on ND will help to identify the protein-protein and protein-membrane interfaces critical for the Tenase complex assembly and function.