Abstract
Heterotrimeric G proteins (Gα, Gβ and Gγ) act downstream of G-protein-coupled receptors (GPCRs) to mediate signaling pathways that regulate various physiological processes and human disease conditions. While human Gαi and its yeast homolog Gpa1 were previously postulated to function as intracellular pH sensors, the pH-sensing capabilities of Gαi and the underlying mechanism remain to be established. Our research shows that variations in pH significantly affect the structure and stability of Gαi-GDP. Specifically, at the lower end of the physiological pH range, the protein undergoes an order-to-disorder transition due to the loss of electrostatic interactions within the Gαi Switch regions, resulting in a reduction in agonist-mediated Gαi-Gβγ release. Further, we identified key residues within the Gαi Switch regions that form the pH-sensing network. Mutation of these residues in Gαi gives rise to 'low pH mimetics' that abolish pH-dependent thermostability changes and reduce Gαi-Gβγ release. Overall, our findings suggest that pH-sensitive structural changes in Gαi impact the agonist-mediated dissociation of Gβγ, which is essential for proper signaling.