Intrinsic GTPase Activity of K-RAS Monitored by Native Mass Spectrometry

Abstract

Mutations in RAS are associated with many different cancers and have been a therapeutic target for more than three decades. RAS cycles from an active to inactive state by both intrinsic and GTPase-activating protein (GAP)-stimulated hydrolysis. The activated enzyme interacts with downstream effectors, leading to tumor proliferation. Mutations in RAS associated with cancer are insensitive to GAP, and the rate of inactivation is limited to their intrinsic hydrolysis rate. Here, we use high-resolution native mass spectrometry (MS) to determine the kinetics and transition state thermodynamics of intrinsic hydrolysis for K-RAS and its oncogenic mutants. MS data reveal heterogeneity where both 2′-deoxy and 2′-hydroxy forms of GDP (guanosine diphosphate) and GTP (guanosine triphosphate) are bound to the recombinant enzyme. Intrinsic GTPase activity is directly monitored by the loss in mass of K-RAS bound to GTP, which corresponds to the release of phosphate. The rates determined from MS are in direct agreement with those measured using an established solution-based assay. Our results show that the transition state thermodynamics for the intrinsic GTPase activity of K-RAS is both enthalpically and entropically unfavorable. The oncogenic mutants G12C, Q61H, and G13D unexpectedly exhibit a 2′-deoxy GTP intrinsic hydrolysis rate higher than that for GTP.

Overcoming Resistance to Drugs Targeting KRAS Mutation

“… 37 , 38 Upon G12 and G13 mutation, GAPs-mediated K-Ras inactivation is hampered, resulting in the accumulation of GTP-bound active K-Ras. 24 , 39 …”

K-Ras prenylation as a potential anticancer target

“…In addition, mutational patterns may also modulate tissue-specific differences. For example, KRAS G12C, G12V, G13D, or Q61L exhibit differential intrinsic GTPase activity, sensitivity to GAP-mediated inactivation, or activation by GEF proteins [ 57 , 58 ]. Thus, distinct properties of the different mutations along with tissue-specific mutational patterns may mediate differential sensitivity to prenylation inhibitors.…”

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

“…Other soluble proteins are more closely associated with the membrane, like K-Ras, which is stable in solution but remains anchored to the membrane via palmitoylated cysteine residue. To facilitate nMS analysis, Laganowsky and co-workers therefore used a C-terminally truncated version lacking the last 19 amino acids including the farnesylation site at Cys180 [38]. With this modification, the authors were able to preserve the interactions between K-Ras and nucleotides under native MS conditions.…”

“…With this modification, the authors were able to preserve the interactions between K-Ras and nucleotides under native MS conditions. By monitoring the conversion of GTP to GDP in the intact protein complex, they could determine kinetic parameters for GTP hydrolysis and map the impact of oncogenic mutations on the GTPase activity of K-Ras [38].…”