Exploring binding and allosteric energy landscapes for the KRAS interactions with effector proteins using Markov state modeling of conformational ensembles and allosteric network modeling

Kirsten rat sarcoma viral oncogene homolog (KRAS) is a pivotal oncoprotein that regulates cell proliferation and survival through interactions with downstream effectors such as RAF1. Despite significant advances, the dynamic and energetic mechanisms of KRAS allostery by which oncogenic mutations can modulate KRAS-RAF1 signaling remain poorly understood. In this study, we employ microsecond molecular dynamics simulations, mutational scanning, and binding free energy calculations together with dynamic network modeling to elucidate the effect of KRAS G12V, G13D, and Q61R mutations and characterize the thermodynamic drivers and hotspots of KRAS binding and allostery. We found that these mutations stabilize the active state and enhance RAF1 binding by differentially modulating the flexibility of switch regions. The G12V mutation rigidifies both switch I and switch II, locking KRAS in a stable active state. In contrast, the G13D mutation moderately reduces switch I flexibility, while the Q61R mutation induces a more dynamic conformational landscape. Mutational scanning and binding free energy analysis of KRAS-RAF1 complexes identified key binding affinity hotspots that leverage synergistic electrostatic and hydrophobic binding interactions in stabilizing the KRAS-RAF1 interfaces. Dynamic network analysis identifies critical allosteric centers and a conserved allosteric architecture that mediate long-range interactions in the KRAS-RAF1 complexes and enable precision modulation of KRAS dynamics in oncogenic contexts. The predictions accurately reproduced the experimental data on KRAS allostery and provided a detailed map of allosteric communications mediated by the central β-sheet region of KRAS that connects the binding interface hotspots with allosteric hubs transmitting functional conformational changes. Together, these findings advance our understanding of mechanisms underlying allosteric regulation of KRAS binding and underscore the importance of targeting mutant-specific conformations for therapeutic interventions.