G12 mutations rewire allosteric communication at the Ras-RalGDS interface

The Ras subfamily is the most extensively studied branch of the Ras superfamily, with 20% of all human tumors having activating mutations in one of the RAS genes. Recent studies have shown that the Ras/RalGDS/Ral pathway plays a more significant role in the progression of Ras-driven colon and pancreatic cancers than the Ras/Raf and Ras/PI3K pathways. In this study, we investigated the interaction between Ras and the Ras/Rap binding domain (RBD) of RalGDS using long-timescale molecular dynamics simulations. The binding free energy of dimerization showed that Rap1-RBD has the strongest interaction and M-Ras-RBD the weakest interaction among the simulated systems, consistent with experimental results. We noticed that Ras uses the same acidic interface residues when binding to the complementary basic residues of RalGDS and Raf. By analyzing bonding profiles, we identified several conserved interactions across different systems as well as isoform- and mutant-specific preferences. Our results demonstrate that G12D/V mutations favor Glu37-mediated stabilization, specifically through the Glu37^Ras-Ser817^RBD hydrogen bond and the Glu37^Ras-Tyr815^RBD anion-π interaction. By mapping interface allosteric communication pathways, we illustrated the interplay between these stabilizing interactions and allosteric signal transduction across the dimer. We hypothesize that communication between the Ras active site and the RalGDS RBD is rewired upon G12 mutations. Specifically, we identified the GTP-Gly/Asp/Val12^Ras-Gln61^Ras-Tyr64^Ras-Ile36^Ras-Ile803^RBD pathway that exhibits divergent behavior in wild-type versus mutant systems. The interaction dynamics represented here may serve as a good reference point for studies aiming to develop mutant-specific targeting against tumors harboring Ral overactivity.