Uncovering a membrane-distal conformation of KRAS available to recruit RAF to the plasma membrane

Significance The proto-oncogene KRAS, a small GTPase, is frequently mutated in pancreatic, colorectal, and lung cancer. These mutations result in elevated levels of the activated guanosine triphosphate-bound form of KRAS. Localized at the plasma membrane, KRAS functions to recruit effectors, predominantly RAF kinase for activation and initiation of the MAPK signaling cascade. Combining computational and biophysical methods we identify a membrane-distal state of the KRAS G-domain that alternates with two previously described membrane-proximal states through dynamic reorganization of the hypervariable region. Comprising about 90% of the ensemble, this membrane-distal state of the G-domain dominates the proximal states and may facilitate KRAS to recruit cytosolic RAF kinase to the membrane by a fly-casting mechanism. The small GTPase KRAS is localized at the plasma membrane where it functions as a molecular switch, coupling extracellular growth factor stimulation to intracellular signaling networks. In this process, KRAS recruits effectors, such as RAF kinase, to the plasma membrane where they are activated by a series of complex molecular steps. Defining the membrane-bound state of KRAS is fundamental to understanding the activation of RAF kinase and in evaluating novel therapeutic opportunities for the inhibition of oncogenic KRAS-mediated signaling. We combined multiple biophysical measurements and computational methodologies to generate a consensus model for authentically processed, membrane-anchored KRAS. In contrast to the two membrane-proximal conformations previously reported, we identify a third significantly populated state using a combination of neutron reflectivity, fast photochemical oxidation of proteins (FPOP), and NMR. In this highly populated state, which we refer to as “membrane-distal” and estimate to comprise ∼90% of the ensemble, the G-domain does not directly contact the membrane but is tethered via its C-terminal hypervariable region and carboxymethylated farnesyl moiety, as shown by FPOP. Subsequent interaction of the RAF1 RAS binding domain with KRAS does not significantly change G-domain configurations on the membrane but affects their relative populations. Overall, our results are consistent with a directional fly-casting mechanism for KRAS, in which the membrane-distal state of the G-domain can effectively recruit RAF kinase from the cytoplasm for activation at the membrane.