A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα<sub>o</sub><sup>K46E</sup> has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα<sub>o</sub><sup>K46E</sup> forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα<sub>o</sub>, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

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Publication Details

Journal
Nature Communications
Published
2024-08-05
DOI
https://doi.org/10.1038/s41467-024-50964-z
Citations
15
Primary Topic
Receptor Mechanisms and Signaling
Type
article
Field-Weighted Citation Impact
3.51

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article

A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state

Alan V. Smrcka, Bryan L. Roth, Diomedes E. Logothetis, Nicholas J. Kapolka et al.
15 citations
Nature Communications
Receptor Mechanisms and Signaling
3.51
article

A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state

Alan V. Smrcka, Bryan L. Roth, Diomedes E. Logothetis, Nicholas J. Kapolka, Kevin M. Knight, Henrik Dohlman, Wenyuan Wei, W.H. Ludlam, Nagarajan Vaidehi, Kirill A. Martemyanov, Iya Prytkova, Jonathan F. Fay, Ning Ma, Tyler J. Lefevre, B. Krumm, Paul A. Slesinger, Meng Cui, Xi‐Ping Huang, Elizabeth G. Obarow, Sepehr Mani
article en
15 citations

Abstract

Many neurotransmitter receptors activate G proteins through exchange of GDP for GTP. The intermediate nucleotide-free state has eluded characterization, due largely to its inherent instability. Here we characterize a G protein variant associated with a rare neurological disorder in humans. Gα<sub>o</sub><sup>K46E</sup> has a charge reversal that clashes with the phosphate groups of GDP and GTP. As anticipated, the purified protein binds poorly to guanine nucleotides yet retains wild-type affinity for G protein βγ subunits. In cells with physiological concentrations of nucleotide, Gα<sub>o</sub><sup>K46E</sup> forms a stable complex with receptors and Gβγ, impeding effector activation. Further, we demonstrate that the mutant can be easily purified in complex with dopamine-bound D2 receptors, and use cryo-electron microscopy to determine the structure, including both domains of Gα<sub>o</sub>, without nucleotide or stabilizing nanobodies. These findings reveal the molecular basis for the first committed step of G protein activation, establish a mechanistic basis for a neurological disorder, provide a simplified strategy to determine receptor-G protein structures, and a method to detect high affinity agonist binding in cells.

Nature CommunicationsVol. 15(1)
University of North Carolina at Chapel Hill (US), University of Maryland, Baltimore (US), Northeastern University (US), University of North Carolina Health Care (US), University of Michigan (US), University of Florida (US), City of Hope (US), Beckman Research Institute, Icahn School of Medicine at Mount Sinai (US)
National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, National Heart, Lung, and Blood Institute, National Institute of Mental Health, National Institute on Drug Abuse, National Institute of General Medical Sciences, National Institute of Neurological Disorders and Stroke, U.S. National Library of Medicine
Openalex Percentile: Top 7%
Receptor Mechanisms and Signaling
3.51
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