Abstract A047: KRAS G12C and G12D mutants exhibit distinct conformational flexibility in the helix 3–switch 2 pocket that drives differential protein function

Abstract KRAS oncogenic mutations, including the common G12C and G12D variants, impair GTP hydrolysis and occur at high frequency in cancer. The unexplained variations in RAS mutant allele frequency and tissue distribution, and the question of whether mutant-specific preferences for effector binding contribute to their prevalence in cancer, remain unanswered. Our NMR solution structures reveal that the SW2-pocket (SIIP) is larger in KRASG12D than in KRASG12C. This difference arises from distinct conformations of the P-loop, the SW2 loop, and the N-terminal a2 helix that are coordinated by perturbations in SW1 region. Codon 12 in the P-loop and residue Q61 within SW2 are key contributors to these structural differences. KRASG12D shows multiple D12 rotamers, whereas KRASG12C adopts a single C12 rotamer that restricts access to the nucleotide gamma-phosphate (gP) which imposes constraints on SIIP-directed inhibitor design. The Q61 side chain also adopts mutant-specific packing: in KRASG12C the side chain is constrained by interaction with Y96 leading to a narrowed pocket compared to that in KRASG12D where it shifts away from the SIIP, increasing its distance from the gP. These distinctions suggest that the mutants alter the catalytic environment for GTP hydrolysis. Our analysis of protein dynamics by NMR supports unique SW2 packing observed in KRASG12D. We identify a potent pan-KRAS small-molecule inhibitor, BBO-11534, that binds both GTP- and GDP-loaded KRAS. 31P NMR analyses demonstrate that BBO-11534 shifts the conformational equilibrium of GTP-bound KRASWT and KRASG12D toward the signaling-incompetent state 1. In contrast, this shift is modest in GTP-KRASG12C, likely due to confinement of the restricted C12 rotamer and smaller SIIP, which limits inhibitor access to the gP. This distinct conformational network within the active site provides mechanistic rationale for these allele-specific responses. To assess the impact of these structural differences between KRASG12D vs KRASG12C, we examined PI3K p110α binding in cells using a Bioluminescence Resonance Energy Transfer (BRET) assay. These measurements show that KRASG12D binds p110α with the highest affinity, followed by KRASG12C and then KRASWT. Chemical shift perturbation NMR and molecular dynamics simulations recapitulate this binding hierarchy (G12D > G12C > WT). Likewise, immunoprecipitation of FLAG-tagged KRASG12D in HEK293T cells captures more PI3Kα than the KRASG12C or KRASWT proteins, further supporting this trend. Together, this study identifies unique structural features in oncogenic KRAS mutants that demonstrate how these features influence protein function and provide guidance for the design of allele-specific therapeutics. Citation Format: ALOK K. SHARMA, Megan Rigby, MARCO TONELLI, Nicole FER, Patrick Alexander, JUN PEI, YUE YANG, DANA Rabara, Erik K. Larsen, Brian P. Smith, MA ROGER, Vandana Kumari, Marcin Dyba, Felice Lightstone, BIN WANG, PEDRO J. BELTRAN, Eli Wallace, Andrew G. Stephen, Dwight V. Nissley, Frank McCormick, Anna E. Maciag. KRAS G12C and G12D mutants exhibit distinct conformational flexibility in the helix 3–switch 2 pocket that drives differential protein function [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RAS Oncogenesis and Therapeutics; 2026 Mar 5-8; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_1):Abstract nr A047.