A Conserved Phenylalanine as a Relay between the α5 Helix and the GDP Binding Region of Heterotrimeric Gi Protein α Subunit

Background: GPCRs regulate heterotrimeric G protein activation. However, the intermediate steps regulating GDP release are still unknown. Results: Energy analysis pinpoints information flow through G-receptor interaction and GDP release. Conclusion: Hydrophobic interactions around 5 helix, 2-3 strands, and 1 helix are key for GDP stability. Significance: G protein activation defines regulation of high affinity receptor interactions and plays a role defining different cellular responses. G protein activation by G protein-coupled receptors is one of the critical steps for many cellular signal transduction pathways. Previously, we and other groups reported that the 5 helix in the G protein subunit plays a major role during this activation process. However, the precise signaling pathway between the 5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the 5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the 2-3 strands and 1 helix, leading to high basal nucleotide exchange. However, mutations in strands 5 and 6 do not perturb G protein activation. We have highlighted critical residues that leverage Phe-336 as a relay. Conformational changes are transmitted starting from Phe-336 via 2-3/1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the 1 and 5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal 5 helix, mutation of Phe-336 still leads to high basal exchange rates. This suggests that unlike receptor-mediated activation, helix 5 rotation and translocation are not necessary for GDP release from the subunit. Rather, destabilization of the backdoor region of the G subunit is sufficient for triggering the activation process.