4PAO: A conserved phenylalanine as relay between the 5 helix and the GDP binding region of heterotrimeric G protein

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 alpha5 helix in the G protein alpha subunit plays a major role during this activation process. However, the precise signaling pathway between the alpha5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the alpha5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the beta2-beta3 strands and alpha1 helix, leading to high basal nucleotide exchange. However, mutations in beta strands beta5 and beta6 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 beta2-beta3/alpha1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the alpha1 and alpha5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal alpha5 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 alpha subunit. Rather, destabilization of the backdoor region of the Galpha subunit is sufficient for triggering the activation process.
PDB ID: 4PAODownload
MMDB ID: 121940
PDB Deposition Date: 2014/4/9
Updated in MMDB: 2017/12
Experimental Method:
x-ray diffraction
Resolution: 2.003  Å
Source Organism:
Similar Structures:
Biological Unit for 4PAO: monomeric; determined by author and by software (PISA)
Molecular Components in 4PAO
Label Count Molecule
Protein (1 molecule)
Guanine Nucleotide-binding Protein G(i) Subunit Alpha-1(Gene symbol: Gnai1)
Molecule annotation
Chemicals (2 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB