Structural Dynamics in Ras and Related Proteins upon Nucleotide Switching

Abstract

Structural dynamics of Ras proteins contribute to their activity in signal transduction cascades. Directly targeting Ras proteins with small molecules may rely on movement of a conserved structural motif, switch II. To understand Ras signaling and advance Ras targeting strategies, experimental methods to measure Ras dynamics are required. Here we demonstrate the utility of hydrogen-deuterium exchange mass spectrometry to measure Ras dynamics by studying representatives from two branches of the Ras superfamily, Ras and Rho. A comparison of differential deuterium exchange between active (GMPPNP-bound) and inactive (GDP-bound) proteins revealed differences between the families, with the most notable differences occurring in the phosphate-binding loop and switch II. The P-loop exchange signature correlated with switch II dynamics observed in molecular dynamics simulations focused on measuring main chain movement. Hydrogen-deuterium exchange provides a means of evaluating Ras protein dynamics which may be useful for understanding mechanisms of Ras signaling, including activated signaling of pathologic mutants, and for targeting strategies that rely on protein dynamics.

Novel K-Ras G12C Switch-II Covalent Binders Destabilize Ras and Accelerate Nucleotide Exchange

“…HDX-MS data of K-Ras G12C bound to GDP were consistent with previously published reports, confirming the flexibility of both the switch I and switch II loops. 32 …”

KRAS G12C Drug Development: Discrimination between Switch II Pocket Configurations Using Hydrogen/Deuterium-Exchange Mass Spectrometry

“…HDX MS experiments were performed in a similar manner to those described previously (Harrison et al, 2016;Lim et al, 2014). Samples containing KRAS G12C alone and bound to each compound (1 and ARS) were independently labeled with deuterium in triplicate, using identical experimental conditions so that each compound-bound form could be compared to KRAS G12C alone.…”

“…The labeling reaction was quenched at five predetermined time points (10 s, 1 m, 10 m, 1 h, 4 h) through the addition of 64.0 mL quench buffer (2.0 M guanidinium chloride, 0.8% formic acid, pH 2.1) at 0 C. Quenched samples were immediately flash frozen using dry ice and were stored at -80 C for less than one week prior to analysis. Deuterium measurement with mass spectrometry was performed as previously described (Harrison et al, 2016;Lim et al, 2014). Deuterium incorporation graphs ( Figure S5) were generated using DynamX 3.0 software (Waters) by subtracting the centroid of the isotopic distribution at each labeling time point from the centroid of the isotopic distribution of the undeuterated reference species.…”

“…We previously used HDX MS to differentiate between guanosine monophosphate-purine nucleoside phosphorylase-and GDP-bound KRAS G12C, and showed that the protein dynamics of KRAS G12C covalently bound to GDP-mimetic SML-8-73-1 are similar to the inactive, GDPbound protein (Lim et al, 2014). In another study, we used HDX MS to elucidate differences in switch II behavior that distinguish members of the Ras subfamily from the Rho subfamily (Harrison et al, 2016). Given the ability of HDX MS to detect changes in switch II dynamics, we hypothesized that HDX MS would be able to detect different conformations of switch II induced by the two major classes of SIIP binders, if such differences exist.…”

“…The flexibility and conformational dynamics of switch II complicates structure-guided drug design work on quinazoline scaffolds because the conformation of switch II is uncertain. Here we demonstrate that hydrogen/deuterium-exchange mass spectrometry (HDX MS), a method capable of detecting differences in switch II dynamics for native RAS family proteins (Harrison et al, 2016), can detect differences in the conformational state of switch II induced by the quinazoline versus chloro hydroxy aniline scaffolds. Further, we use X-ray crystallography to explain the differences in HDX MS signatures seen after treatment with quinazoline-containing compounds.…”