Abstract
Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. Numerous GTPases are implicated in oncogenesis, particularly the three RAS isoforms HRAS, KRAS, and NRAS and the RHO family GTPase RAC1. Signaling networks comprising small GTPases are highly connected, and there is some evidence of direct biochemical cross-talk between their functional G-domains. The activation potential of a given GTPase is contingent on a codependent interaction with the nucleotide and a Mg2+ ion, which bind to individual variants with distinct affinities coordinated by residues in the GTPase nucleotide-binding pocket. Here, we utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system. We provide insight into nucleotide preference and the role of Mg2+ in activating both WT and oncogenic mutant enzymes. Multiplexing revealed guanine nucleotide exchange factor (GEF), GTPase-activating protein (GAP), and effector-binding specificities in mixtures of GTPases and resolved that the three related RAS isoforms are biochemically equivalent. This work establishes that direct quantitation of the nucleotide-bound conformation is required to accurately determine an activation potential for any given GTPase, as small GTPases such as RAS-like proto-oncogene A (RALA) or the G12C mutant of KRAS display fast exchange kinetics but have a high affinity for GDP. Furthermore, we propose that the G-domains of small GTPases behave autonomously in solution and that nucleotide cycling proceeds independently of protein concentration but is highly impacted by Mg2+ abundance.
Highlights
Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes
We utilized a selective-labeling strategy coupled with real-time NMR spectroscopy to monitor nucleotide exchange, GTP hydrolysis, and effector interactions of multiple small GTPases in a single complex system
Nucleotide exchange and GTP hydrolysis occur intrinsically; exchange and hydrolysis rates are catalyzed by guanine nucleotide exchange factors (GEFs)3 or GTPase-activating proteins (GAPs), respectively
Summary
Small GTPases alternatively bind GDP/GTP guanine nucleotides to gate signaling pathways that direct most cellular processes. To accurately quantify the activation state of GTPases requires consideration of relative nucleotide affinity (i.e. preference of a given GTPase to bind GDP or GTP), the availability of Mg2ϩ cofactor, and the potential impact of multimer formation or membrane interactions This would take into consideration the growing evidence that RAS GTPases dimerize [18, 19], which would be intensified at high protein concentrations such as those in membrane nanoclusters. To strengthen the RT-NMR approach, it is possible to multiplex these assays [23], allowing quantification of activation states for several GTPases monitored simultaneously in real time This approach could resolve whether GTPases behave autonomously in mixtures or whether they are highly interactive with each other or at concentrations that promote oligomerization and whether this impacts nucleotide exchange or GTP hydrolysis kinetics and/or effector binding. The data improve our understanding of the complexity and interconnectedness of small GTPases in the context of cell signaling, the impact of relative nucleotide affinity, Mg2ϩ availability, and cross-talk mechanisms
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