Positive cooperativity between RAS-binding and cysteine-rich domains regulates RAF membrane binding kinetics via lateral rebinding

RAF activation requires coordinated interactions with both RAS and membrane lipids, yet the molecular basis of this process remains unclear. Using a bottom-up reconstitution approach, we show how coordinated protein-protein and protein-lipid interactions regulate membrane binding dynamics of RAF to drive its multistep activation. Within membrane environments, the RAS-binding domain (RBD) and cysteine-rich domain (CRD) exhibit cooperativity, with CRD-mediated phosphatidylserine binding stabilizing the RBD:RAS complex. Importantly, RAF remains membrane-bound through lateral rebinding to RAS, where a weak CRD-lipid interaction plays an essential role. The lateral rebinding extends RAF's membrane dwell time under high RAS density conditions. This prolonged membrane residence may facilitate completion of RAF's multistep activation. Given the high abundance of weak multivalent membrane interactions, lateral rebinding may be a common mechanism for regulating the activity of signaling proteins through sustained membrane retention.