Magnesium as a conformational gatekeeper of KRAS: Structural dynamics and therapeutic implications

Magnesium serves as an essential cofactor for small GTPases, yet its structural role in regulating KRAS conformational dynamics and nucleotide exchange remains poorly understood. Here, we combine hydrogen-deuterium exchange mass spectrometry (HDX-MS), native mass spectrometry, and functional assays to elucidate how Mg²⁺ stabilizes the KRAS conformational ensemble and constrains transitions between GDP- and GTP-bound states. Depletion of Mg²⁺ triggers widespread increases in structural dynamics throughout KRAS-spanning the p-loop, α1-helix, switch I, nucleotide-binding region, and distal helices-revealing a global loosening of the protein fold that favors an open, nucleotide exchange-competent state. Mg²⁺ titration experiments demonstrate that individual structural elements exhibit distinct Mg²⁺ dependencies: the p-loop and α1-helix recover native dynamics at micromolar concentrations, whereas switch I requires millimolar levels, underscoring its exceptionally high sensitivity to Mg²⁺ for structural stabilization. KRAS bound to the catalytic domain of exchange factor SOS1 displays an HDX signature closely resembling the Mg²⁺-free state, indicating that SOS1 promotes nucleotide exchange by transiently perturbing Mg²⁺ coordination while simultaneously stabilizing switch I. Consistently, the phosphomimetic KRAS S17E variant, which disrupts a critical Mg²⁺-coordinating residue, exhibits pronounced global destabilization-reinforcing the central importance of Mg²⁺ in maintaining structural integrity. Taken together, our findings show that Mg²⁺ acts as a master regulator of KRAS structural dynamics and reveal Mg²⁺-sensitive hotspots that might represent promising targets for next-generation KRAS therapeutics.