Organization, dynamics, and segregation of Ras nanoclusters in membrane domains

Recent experiments have shown that membrane-bound Ras proteins form transient, nanoscale signaling platforms that play a crucial role in high-fidelity signal transmission. However, a detailed characterization of these dynamic proteolipid substructures by high-resolution experimental techniques remains elusive. Here we use extensive semiatomic simulations to reveal the molecular basis for the formation and domain-specific distribution of Ras nanoclusters. As model systems, we chose the triply lipidated membrane targeting motif of H-ras (tH) and a large bilayer made up of di16:0-PC (DPPC), di18:2-PC (DLiPC), and cholesterol. We found that 4–10 tH molecules assemble into clusters that undergo molecular exchange in the sub-μs to μs time scale, depending on the simulation temperature and hence the stability of lipid domains. Driven by the opposite preference of tH palmitoyls and farnesyl for ordered and disordered membrane domains, clustered tH molecules segregate to the boundary of lipid domains. Additionally, a systematic analysis of depalmitoylated and defarnesylated tH variants allowed us to decipher the role of individual lipid modifications in domain-specific nanocluster localization and thereby explain why homologous Ras isoforms form nonoverlapping nanoclusters. Moreover, the localization of tH nanoclusters at domain boundaries resulted in a significantly lower line tension and increased membrane curvature. Taken together, these results provide a unique mechanistic insight into how protein assembly promoted by lipid-modification modulates bilayer shape to generate functional signaling platforms.