Quantitative Measurement of Intrinsic GTP Hydrolysis for Carcinogenic Glutamine 61 Mutants in H-Ras

Abstract

Mutations of human oncoprotein p21H-Ras (hereafter “Ras”) at glutamine 61 are known to slow the rate of guanosine triphosphate (GTP) hydrolysis and transform healthy cells into malignant cells. It has been hypothesized that this glutamine plays a role in the intrinsic mechanism of GTP hydrolysis by interacting with an active site water molecule that stabilizes the formation of the charged transition state at the γ-phosphate during hydrolysis. However, there is no comprehensive data set of the effects of mutations to Q61 on the protein’s intrinsic catalytic rate, structure, or interactions with water at the active site. Here, we present the first comprehensive and quantitative set of initial rates of intrinsic hydrolysis for all stable variants of RasQ61X. We further conducted enhanced molecular dynamics (MD) simulations of each construct to determine the solvent accessible surface area (SASA) of the side chain at position 61 and compared these results to previously measured changes in electric fields caused by RasQ61X mutations. For polar and negatively charged residues, we found that the rates are normally distributed about an optimal electrostatic contribution, close to that of the native Q61 residue, and the rates are strongly correlated to the number of waters in the active site. Together, these results support a mechanism of GTP hydrolysis in which Q61 stabilizes a transient hydronium ion, which then stabilizes the transition state while the γ-phosphate is undergoing nucleophilic attack by a second, catalytically active water molecule. We discuss the implications of such a mechanism on future strategies for combating Ras-based cancers.

KRASQ61H Preferentially Signals through MAPK in a RAF Dimer-Dependent Manner in Non–Small Cell Lung Cancer

“…S3B). Of note, bridging waters, which normally play a role in catalysis, are key components of these interactions (31). With the mutation of Gln to His at position 61, the side chain no longer interacts with a key bridging water, leading to a breakdown in the remaining network and an unconstrained switch 2 (Fig.…”

Crystal structure and function of Rbj: A constitutively GTP-bound small G protein with an extra DnaJ domain

“…2D). Previous studies indicated that Q61 in the switch II loop of Ras plays a key role in the intrinsic GTP hydrolysis reaction (Novelli et al, 2018) (Fig. 2A).…”

Functional differences drive the selection of NRAS mutants in melanoma

“…Codon 12 and 13 mutants prevent GAP-mediated catalysis of GTP hydrolysis (Adari et al 1988), whereas codon 61 alterations abolish intrinsic RAS GTPase activity (Frech et al 1994). On the other hand, minimal differences are observed in the GTP hydrolysis rates between RAS codon 61 mutants (Novelli et al 2018),raising the question of why certain codon 61 mutants would be more prevalent in melanoma. We explored this question using a new suite of melanoma GEMMs that enable the conditional and melanocyte-specific expression of eight different NRAS mutants from the endogenous gene locus.…”

“…Emerging evidence shows that RAS mutants have distinct biochemical and tumorigenic properties. While all oncogenic RAS mutants are constitutively active, differential positioning of the switch I and II domains leads to variances in GTP binding and hydrolysis (Lu et al 2016;Novelli et al 2018). These structural differences can also influence effector interactions (Céspedes et al 2006;Buhrman et al 2007;Stolze et al 2014;Hunter et al 2015; Marcus and Mattos 2015) as evidenced by the positioning of switch II in KRAS 12R , which prevents PI3Kα binding and the subsequent induction of macropinocytosis (Hobbs et al 2020).…”