A Molecular Dynamics Protocol for Predicting EGFR Overactivation and Its Application to the Allosteric Mutations S768I, S768N, and D761N

Aberrant signaling due to mutations in the Epidermal Growth Factor Receptor (EGFR) kinase domain is implicated in various diseases, including cancer. However, the structural mechanisms underlying overactivation in many rare EGFR mutations remain poorly understood. Here, we benchmarked the CHARMM and AMBER force fields and compared simulations of asymmetric and symmetric dimers to establish a Molecular Dynamics (MD) protocol capable of revealing EGFR mutant actional modes using relatively short simulations. This protocol successfully reproduced the known mechanistic behavior of wild-type EGFR and two common mutations, L858R and T790M/L858R. We then applied it to three rare allosteric EGFR mutations: S768I, S768N, and D761N. Experimental studies have suggested that S768I and D761N are oncogenic, whereas S768N is likely a neutral mutation that does not significantly alter EGFR activity. Our simulations showed that S768I and S768N affect the orientation and stability of the catalytically important αC-helix allosterically, while D761N introduces a new hydrogen bonding network between the αC-helix and activation loop. These findings demonstrate how mutations induce distinct allosteric effects that alter EGFR dynamics and potentially contribute to overactivation. The benchmarking protocol established in this study provides a robust framework for investigating EGFR mutations and is readily applicable to additional variants.