KRAS4b functions as a molecular switch cycling between an active GTP bound state and inactive GDP state. Active KRAS4b binds tightly to RAF1 via its Ras binding domain (RBD), however activation of RAF1 competent for MAPK signal transduction requires membrane bound KRAS4b. Using recombinant farnesylated and methylated KRAS4b (KRAS4b-FMe) we have used a variety of biophysical methodologies including paramagnetic relaxation enhancement NMR, neutron reflectivity, small angle neutron and x-ray scattering and protein footprinting to gain insights into the orientation and structure of KRAS-FMe bound to a membrane. In these experiments we have used Nanodiscs or tethered lipid bilayers composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline or -serine (70% POPC 30% POPS). Our data indicate that KRAS-FMe is oriented perpendicular to the membrane with minimal direct contacts to the membrane. The nucleotide bound state does not significantly change the orientation relative to the membrane. We will also present data on the effect of the RBD domain of RAF1 binding to KRAS-FMe at the membrane.

Citation Format: Andrew G. Stephen, Que Van, Marco Tonelli, William Gillette, Dominic Esposito, Ben Niu, Michael Gross, Frank Heinrich, Arvind Ramanathan, Christopher Stanley. Structural and biochemical characterization of membrane bound KRAS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5238. doi:10.1158/1538-7445.AM2017-5238