Allosteric Binding-Mediated Suppression on Activity of G12D KRAS Recognized via Markov State Model and Communication Pathway

The KRAS G12D mutation is one of the most common oncogenic lesions in human tumors, especially in pancreatic ductal adenocarcinoma. The monobodies 12D1 and 12D5 exhibit high selectivity for the G12D mutant of KRAS compared to the wild-type (WT) form. However, the structural and dynamic factors underlying this specificity are still not fully understood. To explore this, we analyzed the transition direction of conformations, allosteric communication pathways, and residue-residue interaction networks at the protein-protein interface. The G12D mutation causes the switch regions to transition from a closed state to an open state. Binding of 12D1 and 12D5 restores this abnormal transition. Additionally, the G12D mutation disrupts the regular communication pathway from the allosteric site α3 to the switch regions (SW I and SW II) observed in WT KRAS. Binding of 12D1 and 12D5 to the allosteric site restores this communication pathway to its original state. Detailed protein-protein interaction network analyses further reveal that 12D1 and 12D5 form two specific hydrogen bonds with the backbone carboxylate of D12. These hydrogen bonds not only strengthen the hydrophobic contacts at the monobody-KRAS interface but also correct the abnormal conformational equilibrium and restore the disrupted allosteric circuitry. Overall, our findings confirm that D12 is a structurally and functionally validated anchor for the development of next-generation inhibitors targeting G12D KRAS-driven malignancies.