Structural and Energetic Determinants of Monobody Recognition of Oncogenic KRAS Variants

Two documented clinical failures match this mechanism, or a single Phase 3 failure is on record.

Abstract excerpt

Monobodies are engineered binding proteins that recognize extended protein surfaces and offer advantages over small-molecule inhibitors for targeting challenging KRAS oncoproteins. Monobody 12D4 exhibits high affinity and selectivity for the oncogenic KRAS(G12D) mutant, but the molecular determinants governing its recognition and the basis for its mutant selectivity remain poorly understood. Here, we combined molecular dynamics simulations and energy calculations to characterize the interactions between monobody 12D4 and WT KRAS as well as four clinically relevant oncogenic variants (G12C, G12D, G12V, and G12R) in both GTP- and GDP-bound states. Our simulations revealed that 12D4 recognition depends on a conserved hydrophobic interaction network centered on the monobody FG loop (residues L77, F78, and W79). This network forms stable contacts with KARS Switch II and 3-helix. The energy calculations also showed that residue K75 of 12D4 formed a mutation-specific electrostatic interaction with KRAS G12D. This interaction contributed significantly to the affinity of 12D4 toward this mutant, whereas this interaction was absent in other variants. No monobody currently exists for targeting KRAS G12R in either nucleotide state, and no monobody selectively targets KRAS G12C and G12V in the GDP-bound inactive state. To address these, we performed computational redesign at residues 75. We identified mutations (K75Q, K75Y, and K75M) that enhanced predicted binding to G12C, G12R, and G12V variants through reorganization of interfacial contacts. Our work establishes a structural framework for understanding KRAS-monobody recognition and provides a rational foundation for engineering variant-selective monobodies with improved affinity toward previously untargetable KRAS mutants.