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Conformational resolution of nucleotide cycling and effector interactions for multiple small GTPases determined in parallel.
- Source :
-
The Journal of biological chemistry [J Biol Chem] 2019 Jun 21; Vol. 294 (25), pp. 9937-9948. Date of Electronic Publication: 2019 May 14. - Publication Year :
- 2019
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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 Mg <superscript>2+</superscript> 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 Mg <superscript>2+</superscript> 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 Mg <superscript>2+</superscript> abundance.<br /> (© 2019 Killoran and Smith.)
- Subjects :
- Guanine Nucleotide Exchange Factors chemistry
Humans
Monomeric GTP-Binding Proteins chemistry
Nucleotides chemistry
Protein Binding
Protein Conformation
Proto-Oncogene Mas
Signal Transduction
ras Proteins chemistry
rho GTP-Binding Proteins chemistry
Guanine Nucleotide Exchange Factors metabolism
Monomeric GTP-Binding Proteins metabolism
Nucleotides metabolism
ras Proteins metabolism
rho GTP-Binding Proteins metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1083-351X
- Volume :
- 294
- Issue :
- 25
- Database :
- MEDLINE
- Journal :
- The Journal of biological chemistry
- Publication Type :
- Academic Journal
- Accession number :
- 31088913
- Full Text :
- https://doi.org/10.1074/jbc.RA119.008653