Helical Organization of Coagulation Factor VIII on Lipid Nanotubes

Abstract

Human factor VIII (FVIII) is a multidomain-plasma glycoprotein critical for blood coagulation which when activated (FVIIIa) functions as a co-factor to the serine protease factor IXa (FIXa) within the membrane-bound tenase complex. We have developed single bilayer lipid nanotubes (LNT) resembling by dimensions and lipid composition the activated platelet pseudopodia holding the tenase complex in vivo. We have successfully organized helically recombinant FVIII on LNT and calculate the membrane-bound structure at resolution sufficient to resolve its membrane-bund domain organization. In this work, we present the macromolecular organization of human FVIII helically organized on LNT, as resolved by Cryo-electron microscopy at intermediate resolution (10 - 15 Å) which we compare to the porcine FVIII membrane-bound structure from helical assemblies on LNT at the same experimental conditions. We compare these structures to the FVIII structures when organized in 2D and 3D crystals and when attached to lipid nanodisks (ND), to define the conformational space and flexibility of this important for blood coagulation macromolecule. Our goal is to identify the FVIII protein-protein and protein-lipid interfaces critical for the tenase complex assembly and function. This work is supported by a National Scientist Development grant from the American Heart Association: 10SDG3500034 and UTMB-NCB startup funds to SSM. SSM acknowledge the staff of the Cryo-EM facility, the scientific computing facility and the director of the Sealy Center for Structural biology at UTMB (www.scsb.utmb.edu) Dr. Monte Pettitt.