Authors
Van, Que N. ; Lopez, Cesar A. ; Tonelli, Marco ; Taylor, Troy ; Niu, Ben ; Stanley, Christopher B. ; Bhowmik, Debsindhu; Tran, Timothy H. ; Frank, Peter H. ; Messing, Simon; Alexander, Patrick ; Scott, David; Ye, Xiaoying ; Drew, Matthew ; Chertov, Oleg; Losche, Mathias; Ramanathan, Arvind; Gross, Michael L.; Hengartner, Nicolas W. ; Westler, William M. ; Markley, John L. ; Simanshu, Dhirendra K. ; Nissley, Dwight V. ; Gillette, William; Esposito, Dominic ; McCormick, Frank; Gnanakaran, S. ; Hei
Abstract
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.