Conditional Cooperativity in the RAS Assembly Pathway on Nanodiscs and Altered GTPase Cycling.

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition in the membrane. Notably, the oncogenic mutants assemble in a nonsequential pathways with two mutually cooperative 'α/α' and 'α/β' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/β interaction in a sequential manner. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and to probe their favorable interface interactions. Transiently exposed interfaces are available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain larger numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and to lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.