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

Kevin M. Knight, Brian E. Krumm, Nicholas J. Kapolka, W. Grant Ludlam, Meng Cui, Sepehr Mani, Iya Prytkova, Elizabeth G. Obarow, Tyler J. Lefevre, Wenyuan Wei, Ning Ma, Xi-Ping Huang, Jonathan F. Fay, Nagarajan Vaidehi, Alan V. Smrcka, Paul A. Slesinger, Diomedes E. Logothetis, Kirill A. Martemyanov, Bryan L. Roth and Henrik G. Dohlman ()
Additional contact information
Kevin M. Knight: University of North Carolina at Chapel Hill
Brian E. Krumm: University of North Carolina at Chapel Hill
Nicholas J. Kapolka: University of North Carolina at Chapel Hill
W. Grant Ludlam: University of Florida
Meng Cui: Department of Pharmaceutical Sciences Northeastern University
Sepehr Mani: Department of Pharmaceutical Sciences Northeastern University
Iya Prytkova: Icahn School of Medicine at Mount Sinai
Elizabeth G. Obarow: University of North Carolina at Chapel Hill
Tyler J. Lefevre: University of Michigan
Wenyuan Wei: Beckman Research Institute of the City of Hope
Ning Ma: Beckman Research Institute of the City of Hope
Xi-Ping Huang: University of North Carolina at Chapel Hill
Jonathan F. Fay: Baltimore
Nagarajan Vaidehi: Beckman Research Institute of the City of Hope
Alan V. Smrcka: University of Michigan
Paul A. Slesinger: Icahn School of Medicine at Mount Sinai
Diomedes E. Logothetis: Department of Pharmaceutical Sciences Northeastern University
Kirill A. Martemyanov: University of Florida
Bryan L. Roth: University of North Carolina at Chapel Hill
Henrik G. Dohlman: University of North Carolina at Chapel Hill

Nature Communications, 2024, vol. 15, issue 1, 1-18

Abstract: 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. GαoK46E 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, GαoK46E 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 Gαo, 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.

Date: 2024
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DOI: 10.1038/s41467-024-50964-z

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