Nanoscale studies of protein-membrane interactions in blood clotting

Abstract

Most of the steps in the blood clotting cascade require clotting proteins to bind to membrane surfaces with exposed phosphatidylserine. In spite of the importance of these protein-membrane interactions, we still lack a detailed understanding of how clotting proteins interact with membranes and how membranes contribute so profoundly to catalysis. Our laboratories are using multidisciplinary approaches to explore, at atomic-resolution, how blood clotting protein complexes assemble and function on membrane surfaces.

Fig. 1

Three models for GLA domain binding to a phospholipid bilayer. Residues 1-49 of bovine prothrombin (from PDB file 1NL2 [5]) are shown using a cartoon representation, with the side chains of Gla residues depicted in yellow and Ca²⁺ depicted as purple spheres. The omega loop of the prothrombin GLA domain (which has three exposed hydrophobic residues, Phe5, Leu6 and Val9, whose side chains are not shown in this figure) is: (A) interacting with the headgroup region, tilted ~45° relative to the other models; or (B,C) inserting to varying depths into the bilayer.

Fig. 2

MD simulations of protein-membrane interactions of the fVIIa GLA domain [6]. (A) A snapshot from detailed MD simulations of the fVIIa GLA domain interacting with a PS bilayer. PS molecules whose carboxy and/or phosphate groups are within 5 Å of any Ca²⁺ ions are drawn with a stick representation, with other PS molecules shown as thin lines. Gla residues are coloured in yellow, while the basic side chains of the GLA domain are drawn green. The three hydrophobic residues of the omega loop of the fVIIa GLA domain (Phe4, Leu5, and Leu8) are shown with an orange surface representation. (B) A model for the membrane-associated TF:fVIIa complex built by aligning the fVIIa GLA domain of sTF:fVIIa (from PDB structure 1DAN [39]) to the structure shown in panel A. fVIIa is shown in blue with its active site in red; TF is shown in white. (C) A close up, left-side view depicting a PS molecule tightly bound to the GLA domain, with its headgroup in simultaneous contact with Arg9 and Ca²⁺-8. (D, E) Close-ups of interactions between tightly bound Ca²⁺ ions of the GLA domain and the phosphate moieties of PS molecules. Panels A-C were reprinted from Ohkubo & Tajkhorshid, Distinct structural and adhesive roles of Ca²⁺ in membrane binding of blood coagulation factors. Structure 16:72-81, 2008 [6], with permission from Elsevier.

Fig. 3

Three-dimensional ssNMR (TEDOR) trajectories of the cross-peak observed from the ³¹P of one PS molecule and the ¹⁵N of an adjacent PS molecule [37]. The cross peak intensity as a function of mixing time yields specific internuclear distance information. Also shown, as an insert, is a snapshot taken from MD simulations of Ca²⁺ interacting with PS bilayers, depicting an example of the headgroups of two adjacent PS molecules interacting with the same Ca²⁺ ion. Experimentally determined distances of this type are being used to test structural models of PS headgroup assembly. This figure was reprinted with permission from Biochemistry [37]; copyright 2011 American Chemical Society.