Switch dynamics and activity of GTP-bound HRAS affected by R97 mutations at allosteric helix α3

Allosteric mutations in helix α3 modulate the conformational dynamics and catalytic activity of HRAS; however, the underlying molecular mechanisms remain elusive. Herein, Gaussian accelerated molecular dynamics (GaMD) simulations, Markov state modeling (MSM), and communication network analysis were integrated to elucidate how the R97F, R97G, and R97I mutations at α3 influence HRAS conformational changes. MSM analyses reveal that R97I exerts a more pronounced effect on the conformations of residues I93, K101, D107, L133, and Y137 compared with R97F and R97G. Communication network analysis further demonstrates that all three mutations perturb the communication pathways between α3 and the switch regions, resulting in aberrant signal transduction relative to the wild-type HRAS. Notably, R97I amplifies the structural fluctuations of α3, α4, and loop L8 to a greater extent than R97F and R97G, suggesting a distinct allosteric regulatory mechanism. Moreover, these α3 mutations disrupt the Mg²⁺-mediated interaction network with HRAS and GTP, with R97I inducing markedly different perturbations from those of R97F and R97G. Collectively, these findings provide a mechanistic framework for understanding the functional roles of the RAS family and favor the design of allosteric inhibitors toward HRAS.