Abstract 15108: Structure Determination of Membrane-Bound Human Factor Viii
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. Binding of FVIIIa to FIXa onto the activated platelet surface increase FIXa proteolytic efficiency and thrombin generation more than 100,000 fold, which is critical for normal coagulation. Despite a 4 Å resolution FVIII structure has been solved by X-ray crystallography, the membrane-bound FVIII structure is not yet resolved and the structural basis of the tenase complex assembly and function are not fully understood.
We have developed single bilayer lipid nanotubes (LNT) resembling by dimensions and lipid composition the activated platelet pseudopodia holding the tenase complex in vivo. This functionalized LNT are suitable for helical organization of membrane-associated proteins in close to physiological conditions and We have successfully helically organized recombinant human FVIII bound to LNT, suitable for structural studies by Cryo-electron microscopy at subnanometer resolution (< 1 nm).
In this work, we present the membrane-bound structure of human FVIII, helically organized on lipid nanotubes (LNT) at close to physiological conditions. The FVIII structure at 15 Å resolution was calculated by combining two complimentary Cryo-EM
Methods: single particle and helical reconstruction form 2D images and Cryo-electron tomography (Cryo-ET) from tilted images. To define the flexibility and functional structure of membrane-bound FVIII we have compared the 3D reconstructions and 2D projections obtained with both methods. The resultant human FVIIII-B domain deleted (BDD) membrane-bound structure was compared to the FVIII light-chain and the porcine FVIII-BDD membrane-bound structures from helically organized FVIII molecules on the same LNT.
This knowledge will allow us to define the variation in orientation between closely packed FVIII molecules and understand the structural basis of the protein-membrane and protein-protein interfaces critical for FVIII function and normal hemostasis.
- © 2013 by American Heart Association, Inc.