1H, 15N backbone assignment and comparative analysis of the wild type and G12C, G12D, G12V mutants of K-Ras bound to GDP at physiological pH

Pálfy, Gyula and Vida, István and Perczel, András (2019) 1H, 15N backbone assignment and comparative analysis of the wild type and G12C, G12D, G12V mutants of K-Ras bound to GDP at physiological pH. BIOMOLECULAR NMR ASSIGNMENTS. pp. 1-10. ISSN 1874-2718 (print); 1874-270X (online) (In Press)

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Abstract

K-Ras protein is a membrane-bound small GTPase acting as a molecular switch. It plays a key role in many signal transduction pathways regulating cell proliferation, differentiation, survival, etc. It alternates between its GTP-bound active and the GDP-bound inactive conformers regulated by guanine nucleotide exchange factors and GTPase activating proteins. Its most frequent oncogenic mutants are G12C, G12D, and G12V that have impaired GTPase activity, thus induce malignant tumors. Here we report the resonance assignment of the backbone 1H and 15N nuclei of K-Ras wildtype, G12C, G12D and G12V proteins’ catalytic G domain (1–169 residues) in GDP-bound state, and 13C of backbone and side chains of G12C mutant at physiological pH 7.4. Triple resonance data were used to get secondary structure information and backbone dynamics of G12C, the best-known drug target among K-Ras mutants. Simultaneous investigation of G12C, G12D and G12V mutants, along with the wild type form at the very same conditions allowed us to perform a comprehensive analysis based on the combined chemical shifts to reveal the effect of mutation at G12 position on structure. Intriguingly, the G12C and G12V mutants found to be structurally very similar at the three most important regions of K-Ras (P-loop, Switch-I, Switch-II), while the G12D mutant significantly differs at P-loop and Switch-II from the wildtype as well as G12C and G12V mutants. However, in Switch-I it hardly deviates from the wildtype protein.

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