Towards Piecing Together the RAS-RAF Puzzle: Dynamics of Membrane-Associated KRAS4b-RAF RBD/CRD Ternary Complex

Abstract

The RAS family of small guanine nucleotide-binding proteins are critical for various cellular signaling pathways and are among the most common culprits in human cancer, with KRAS4b mutations occurring at high rates in pancreatic, lung, and colorectal cancer. Membrane-anchored RAS binds to effectors from the RAF kinase family at two domains: (i) the RAS-binding domain (RBD) whose complex with RAS has been solved experimentally, and (ii) the cysteine-rich domain (CRD) that mutational assays show can bind RAS independently. However, the structure of the RAS/RBD/CRD ternary complex still remains unknown, including its membrane orientation and interactions. We have used a combination of all-atom and coarse-grained molecular dynamics (MD) simulations to investigate these questions, focusing on the complex between KRAS4b and the RBD/CRD domains of BRAF that experiments show is the initially-activated RAF isoform. First, single-domain simulations of CRD or RBD alone indicate their associations with the membrane. Next, binary complex simulations based on the RAS/RBD crystal structure show multiple dominant membrane orientations, where RBD could either be away from the membrane or making direct membrane interactions. Finally, extensive simulations based on computational docking of CRD to the RAS/RBD crystal structure suggest a viable ternary complex model where CRD embeds into the membrane via both of its hydrophobic loops, RAS interacts with the membrane at the surface covering helices 4 and 5 of the G domain, and RBD away from the membrane. We are currently investigating if interactions of KRAS4b with the RBD/CRD domains of the CRAF isoform lead to changes in the ternary complex structure and to differential membrane interactions. These studies should provide insights that will contribute to our understanding of both normal and cancer-activated signaling pathways.