The Membrane Tether of the RAS Signaling Protein Drives Nanoclustering by Fly-Casting for Anionic Lipids

Abstract

Cancer is a devastating disease that results from pathological modifications to cellular decision making processes. In healthy cells, the protein-mediated signaling networks that regulate growth and movement are tightly regulated. However, mutations that disrupt or over-activate signaling proteins can drive uncontrolled cell growth. Ras, a peripheral membrane signaling protein, harbors activating mutations in 20% of all cancers, especially those of the pancreas, colon, thyroid, and lung. In theory, the signaling output from overactive Ras mutants can be decreased by small molecules that bind and stabilize inactive states. However, crystallographic structures of Ras reveal a relatively smooth surface lacking substantial hydrophobic crevices for drug binding. Therefore, we use molecular dynamics simulations to explore the topography of Ras proteins and the dynamic accessibility of their effector-binding surfaces. Our long-term goal is the identification of potentially druggable cavities at intermolecular interfaces, including those between Ras and the cell membrane. Our molecular dynamics simulations indicate that, in Ras isoform K-Ras4B, the prenylated 19-residue C-terminal highly variable region (HVR) membrane anchor remains disordered as it pulls Ras' globular G domain closer to membranes enriched in negatively charged (anionic) lipids. Furthermore, we show that although Ras' globular G domain interacts promiscuously with its own HVR and membrane lipids, there are preferential and potentially targetable interactions. Finally, simulations of multiple HVR peptides indicate that intermolecular contacts predominantly involve the G domain-proximal (N-terminal) region of the HVR, consistent with a fly-casting mechanism that augments the self-association of Ras proteins in the context of disordered membrane patches enriched in anionic lipids. This targeted adsorption of the K-Ras4B HVR to anionic membrane surfaces may play a role in regulating the multimerization ability of downstream effector proteins such as Raf kinase.