Journal Article

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

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

2024

Overview

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 o K46E 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 o K46E 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 o , 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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 101/28 / 631/208/737 / 631/378 / 631/45/612/194 / 631/535/1258/1259 / 82/83 / 9/74 / 96/10 / 96/95 / Affinity / Binding / Cell activation / Charge reversal / Cryoelectron Microscopy / Dopamine / Dopamine D2 receptors / Effector cells / Electron microscopy / GTP-Binding Protein alpha Subunits, Gi-Go - chemistry / GTP-Binding Protein alpha Subunits, Gi-Go - genetics / GTP-Binding Protein alpha Subunits, Gi-Go - metabolism / GTP-Binding Protein gamma Subunits - genetics / GTP-Binding Protein gamma Subunits - metabolism / GTP-Binding Proteins - genetics / GTP-Binding Proteins - metabolism / Guanosine Diphosphate - metabolism / Guanosine triphosphate / Guanosine Triphosphate - metabolism / HEK293 Cells / Humanities and Social Sciences / Humans / Molecular structure / multidisciplinary / Mutants / Mutation / Nanobodies / Neurodevelopmental disorders / Neurodevelopmental Disorders - genetics / Neurodevelopmental Disorders - metabolism / Neurological diseases / Neurological disorders / Neurotransmitter receptors / Neurotransmitters / Nucleotides / Protein Binding / Protein structure / Proteins / Receptors / Receptors, Dopamine D2 - genetics / Receptors, Dopamine D2 - metabolism / Science / Science (multidisciplinary)