The Molecular Basis of Cluster Formation by Membrane-Bound Lipidated Ras

Abstract

Recent studies found that stimulus dependent assembly and disassembly of clusters of lipid-anchored signaling proteins on the plasma membrane is a crucial mechanism by which cells achieve high-fidelity signal transmission. The best characterized examples of lipid-protein assemblies are Ras nanoclusters at the inner leaflet of the plasma membrane. Independent of expression level, 30-40% of Ras proteins assemble into 6-8 proteins per cluster. However, little is known about the physical forces underlying the domain-specific localization and clustering of these proteins. To address this issue, we carried out extensive semi-atomistic molecular dynamics (MD) simulations of the C-terminal membrane-targeting motif of H-ras (tH) in a phase-separated bilayer composed of 2000 DPPC, DLiPC, and cholesterol molecules mixed in a 5:3:2 molar ratio. We found that 4-10 tH molecules assemble into dynamic clusters whose stability varies with the extent of lipid phase separation. At ambient temperatures, the calculated cluster size distributions and the clustered fraction agree remarkably well with the available experimental data. Clusters, but not monomers or dimers, segregate to the interface between the liquid ordered and liquid disordered phases. The segregation is driven by the preferential interaction of the saturated palmitoyls in tH clusters with the similarly saturated DPPC, and of the polyunsaturated farnesyl with the unsaturated DLiPC lipids. This was confirmed by additional simulations in which individual lipid modifications were systematically removed through a process of de-palmitoylation and de-farnesylation. The preferential localization of the tH clusters at the domain boundaries resulted in a significant reduction in the line tension and changes in membrane curvature. Initial results on the full-length H-ras suggest that the same fundamental forces drive its clustering, but steric effects modulate the size and distribution of the clusters as well as the elastic properties of the bilayer.