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

Area of Science:

• Biochemistry
• Neuroscience
• Structural Biology

Background:

• G protein-coupled receptors (GPCRs) mediate cellular responses by activating heterotrimeric G proteins.
• G protein activation involves guanine nucleotide exchange (GDP to GTP) on the Gα subunit.
• The intermediate nucleotide-free state is transient and difficult to study.

Purpose of the Study:

• To characterize a G protein variant (Gαo K46E) associated with a neurological disorder.
• To elucidate the structure and function of the nucleotide-free state of G proteins.
• To develop simplified methods for studying GPCR-G protein complexes.

Main Methods:

• Purification and biochemical characterization of the Gαo K46E mutant.
• Cellular assays to assess receptor and G protein interactions.
• Cryo-electron microscopy (cryo-EM) to determine the structure of the Gαo K46E-D2 receptor complex.

Main Results:

• Gαo K46E exhibits poor nucleotide binding but retains affinity for Gβγ subunits.
• The mutant forms stable complexes with receptors and Gβγ in cells, inhibiting effector activation.
• A stable structure of the nucleotide-free Gαo K46E bound to D2 receptor was determined by cryo-EM without nanobodies.

Conclusions:

• The study reveals the molecular basis of the initial step in G protein activation.
• The findings provide a mechanistic understanding of a rare neurological disorder.
• A simplified strategy for determining GPCR-G protein structures and detecting agonist binding was established.