Abstract
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 α5 helix in the G protein α subunit plays a major role during this activation process. However, the precise signaling pathway between the α5 helix and the guanosine diphosphate (GDP) binding pocket remains elusive. Here, using structural, biochemical, and computational techniques, we probed different residues around the α5 helix for their role in signaling. Our data showed that perturbing the Phe-336 residue disturbs hydrophobic interactions with the β2-β3 strands and α1 helix, leading to high basal nucleotide exchange. However, mutations in β strands β5 and β6 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 β2-β3/α1 to Switch I and the phosphate binding loop, decreasing the stability of the GDP binding pocket and triggering nucleotide release. When the α1 and α5 helices were cross-linked, inhibiting the receptor-mediated displacement of the C-terminal α5 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 α subunit. Rather, destabilization of the backdoor region of the Gα subunit is sufficient for triggering the activation process.
Highlights
To test the effect of these residues on G protein function, we evaluated nucleotide exchange rates after introduction of site-directed mutations
The simplest way to explain these data would be that those residues do not play a major role in G protein activation or that a single mutation is not enough to disturb the ␣5 helix for guanosine diphosphate (GDP) release
The results showed that the high nucleotide exchange rates of the mutants could be decreased in elevated Mg2ϩ concentrations (Fig. 6, A and B), suggesting that these mutations had allosteric effects on the phosphate binding region that could be overcome with higher Mg2ϩ concentration
Summary
Dictive computational model of the energy of receptor activation with the goal of understanding conformational changes and connections between potential key residues during G protein activation [26] In this model of the rhodopsin-Gi␣␥ complex, it was suggested that the ␣5 helix is the most critical region for G protein stability and activation and is consistent with previous studies [9, 12, 14, 15, 27]. Energetic analysis predicted that residues Phe-191 and Phe-196 in 2-3, Ile-265 and Phe-267 in 5, Tyr-320 and His-322 in 6 strands, and Gln-52 and Met-53 in the ␣1 helix are making critical interactions with the ␣5 helix in both basal and receptor-mediated G protein activation [26] These key residues might either be important for the overall structural integrity of the GTPase domain during the activation process, or they may be directly involved in activation. With computational analysis provided evidence for a dynamic interplay between Phe-336, the 2 and 3 strands, and the ␣1 helix on the G protein activation route
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