Magnesium acts as KRAS conformational gatekeeper, revealing hotspots for next-generation therapeutics.

The KRAS protein is a pivotal oncogene, with mutations found in approximately 80% of human cancers harboring Ras mutations. Its activity is tightly regulated by cycling between inactive GDP-bound and active GTP-bound states, a process influenced by guanine nucleotide exchange factors (GEFs) like SOS1. Divalent cations, particularly Mg2+, are essential cofactors for small GTPases, yet their precise structural role in governing KRAS conformational dynamics and nucleotide exchange has remained poorly understood, representing a critical gap in developing effective KRAS-targeted therapies.

Researchers employed a combination of hydrogen-deuterium exchange mass spectrometry (HDX-MS), native mass spectrometry, and functional assays to elucidate the role of Mg2+ in KRAS structural dynamics. They performed experiments involving Mg2+ depletion and subsequent Mg2+ titration to observe concentration-dependent effects on KRAS conformation. The study also examined KRAS bound to the catalytic domain of the exchange factor SOS1 and investigated the phosphomimetic KRAS S17E variant, which is known to disrupt a critical Mg2+-coordinating residue, to understand its impact on protein stability and nucleotide exchange.

Depletion of Mg2+ triggered widespread increases in structural dynamics across KRAS, spanning the p-loop, α1-helix, switch I, nucleotide-binding region, and distal helices. This revealed a global loosening of the protein fold that significantly favors an open, nucleotide exchange-competent state. Mg2+ titration experiments demonstrated that individual structural elements exhibit distinct Mg2+ dependencies: the p-loop and α1-helix recovered native dynamics at micromolar concentrations, whereas switch I required millimolar levels, underscoring its exceptionally high sensitivity to Mg2+ for structural stabilization. This differential sensitivity highlights specific regions as potential targets. The phosphomimetic KRAS S17E variant, which disrupts a critical Mg2+-coordinating residue, exhibited pronounced global destabilization, reinforcing the central importance of Mg2+ in maintaining structural integrity. These findings collectively establish Mg2+ as a master regulator of KRAS structural dynamics.

KRAS bound to the catalytic domain of exchange factor SOS1 displayed 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.