A neurodevelopmental disorder mutation locks G proteins in the transitory pre-activated state
Kevin M. Knight, B. Krumm, Nicholas J. Kapolka, W.H. 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, Henrik Dohlman
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. Many neurotransmitters act on receptors coupled to GTP-binding G proteins. Here authors report the structure and activity of a mutant that locks the nucleotide-free and receptor-bound state of the G protein, leading to a rare neurological disorder.