KRAS proteins are localized at the plasma membrane. RAF is complexed with 14-3-3 in an autoinhibited cytoplasmic complex. Binding of the RAS binding domain (RBD) of RAF to KRAS-GTP displaces 14-3-3 and allows the cysteine-rich domain (CRD) to interact with KRAS and the membrane. We investigated the topology of RAF activation by probing the interaction of the RBDCRD domain of RAF to membrane-anchored KRAS-FMe using computational and biophysical methods. Neutron reflectivity measurements of the membrane-bound KRAS-FMe complexed with RBDCRD produced volume occupancy profiles along the membrane normal that closely match structural ensembles of KRAS:RBDCRD obtained from backbone torsion angle Monte Carlo simulations. In these simulations, any membrane interactions except those involving the KRAS C-terminus are ignored, yielding an entropic ensemble. The close match to the experiment provides evidence for a primarily dynamic complex at the membrane. The distances between a pair of spin labels on membrane-bound KRAS-FMe were measured using double electron-electron resonance (DEER), electron paramagnetic resonance (EPR). The DEER distance distributions on KRAS-FMe did not change significantly when bound to RBDCRD, and the Monte Carlo simulations aligned with the DEER EPR distances, further supporting the dynamic nature of the complex. Complementary FRET measurements were performed with Alexa-488 labeled KRAS and a TAMRA-label on the nanodisc belt protein. The change in FRET efficiency with increasing anionic lipid concentration and RBDCRD was investigated. Homogeneous time-resolved fluorescence measurements demonstrated that RBDCRD bound tighter to KRAS-FMe in the presence of nanodiscs in an anionic lipid dependent manner. This result demonstrates that although the overall complex is dynamic, CRD interacts with the membrane in an anionic lipid-dependent manner, contributing to the stability of the membrane-bound structure. Overall, our data support experimentally driven models of membrane-bound KRAS:RBDCRD.
Stephen et al. (Sun,) studied this question.