Impact of KRAS-G12 Mutations on the KRAS-GTP Bound Conformational Dynamics: Structure, Free Energy Barriers and Drugging Pockets

High Resolution Image Download MS PowerPoint Slide KRAS is among the most frequently mutated oncogenes in human cancers, with codon 12 representing a dominant hotspot. Despite decades of study, the atomic-level conformational dynamics of KRAS and its G12 mutations remain insufficiently resolved, posing a central barrier to rational drug discovery. Elucidating these dynamics is critical for revealing hidden druggable pockets and enabling the development of mutation-specific inhibitors. Here, we delineate the impact of KRAS G12 mutations on conformational dynamics and GTP binding by performing microsecond-scale, well-tempered metadynamics simulations. This approach yielded precise free energy landscapes that expose mutation-specific alterations to structural states. By introducing biologically meaningful collective variables, including key angles (ϕ) and distances ( d1, d2 ) defining Switch-I, Switch-II, and the P-loop, we identified distinct local and global minima as well as their transition states. Analysis of these transitions reveals how G12 mutations reshape GTP affinity and drive aberrant KRAS activation. We further demonstrated the convergence of the simulations and discussed the physiological relevance of the calculation results in comparison to previously published experimental and theoretical data. Importantly, our analyses uncovered druggable pockets unique to nonpolar G12 mutants, offering novel entry points for inhibitor design. Collectively, this work provides an atomic-level framework for understanding KRAS G12 mutations, establishes transferable collective variables for broader RAS family studies, and opens new avenues for mutation-specific therapeutic development.