2'-Deoxy Guanosine Nucleotides Alter the Biochemical Properties of Ras

Abstract

Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-Ras^G12C was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOS^cat) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOS^cat differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.

Figure 1.. Recombinant H- and N-Ras proteins co-purify with different guanosine nucleotides.

(A) Native mass spectrum of H-Ras in 200 mM ammonium acetate (pH 7.4). (B) Deconvolution of the mass spectrum shown in panel A. Different nucleotide bound Ras species are labeled. Asterisks denote sodium and/or magnesium bound adducts. (C) Mole fraction of 2’-deoxy guanosine di- and triphosphate bound to H-Ras and oncogenic mutants. (D) Mole fractions of 2’-hydroxy guanosine di- and triphosphate bound to H-Ras and oncogenic mutants. (E) Mole fraction of 2’-deoxy guanosine di- and triphosphate bound to N-Ras and oncogenic mutants. (F) Mole fractions of 2’-hydroxy guanosine di- and triphosphate bound to N-Ras and oncogenic mutants. Reported is the abundance from one measurement.

Figure 2.. Native mass spectra reveal different guanosine nucleotides co-purifiy with K-Ras proteins purified from HEK293 cells.

(A) Deconvolution of K-Ras^WT expressed from HEK293 cells in 200 mM ammonium acetate (pH 7.4). (B) Deconvolution of K-Ras^G12C expressed from HEK293 cells in 200 mM ammonium acetate (pH 7.4). Different nucleotide bound K-Ras species are labeled. Asterisks denote sodium and/or magnesium bound adducts. (C) Mole fraction of 2’-deoxy guanosine di- and triphosphate bound to K-Ras and the oncogenic mutant. (D) Mole fraction of 2’-hydroxy guanosine di- and triphosphate bound to K-Ras and the oncogenic mutant. Reported is the abundance from one measurement.

Figure 3.. Intrinsic GTP and dGTP Hydrolysis rates of H-Ras, N-Ras, and their oncogenic mutants.

A-B) Intrinsic hydrolysis rates of GTP (green) and dGTP (blue) were determined at 25°C for A) H-Ras and B) N-Ras proteins. Reported are the mean and standard deviation (n=3).

Figure 4.. Impact of K-Ras^G12C inhibitors on the intrinsic GTPase activity of K-Ras protein.

(A-B) The percentage of GTP-bound K-Ras proteins plotted over time (dots) and fit to a first-order rate constant model (line) in the presence/absence of K-Ras^G12C inhibitors. (C) Correlation plot of drug bound K-Ras^G12C vs 1-[GTP bound] K-Ras^G12C. R² for linear fit (Sotorasib) is 0.99 and R² for linear fit (Adagrasib) is 0.99. (D) Bar graph of the fold increase in GTP hydrolysis rate. One Asterisk denotes p-values <0.05 and two Asterisks denote p-values <0.01. Reported are the mean and standard deviation (n=3).

Figure 5.. Guanosine nucleotides influence the complexes formed between H-Ras and oncogenic mutants with SOS^cat.

A) Mole fraction of SOS^cat and complexes with H-Ras and oncogenic mutants loaded with GTP. B) Shown as described for C but with H-Ras proteins loaded with dGTP. C) Mole fraction of SOS^cat and complexes with N-Ras and oncogenic mutants loaded with GTP. D) Shown as described for E but with N-Ras proteins loaded with dGTP. Reported are the mean and standard deviation (n=3).