A survey of conformational and energetic changes in G protein signaling

Lokits, Alyssa D.; Leman, Julia Koehler; Kitko, Kristina E.; Hamm, Heidi E.; Meiler, Jens

doi:10.3934/biophy.2015.4.630

Cited by 1 publication

(3 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Briefly, we re-relaxed the previously published ensembles of 10 structures of the G protein in the basal state and receptor-bound state using a DualSpace relax Rosetta protocol (56) for consistency between the mutant structures and the in silico models (55). The G␥ subunit of the receptorbound models was truncated along both the N and C termini to match the available crystal density of the mutant structures.…”

Section: Methodsmentioning

confidence: 99%

“…Rosetta Interface Energy Calculations-Interface energies were computed following the Rosetta ⌬⌬G protocol described previously (54,55). Briefly, we re-relaxed the previously published ensembles of 10 structures of the G protein in the basal state and receptor-bound state using a DualSpace relax Rosetta protocol (56) for consistency between the mutant structures and the in silico models (55).…”

Section: Methodsmentioning

confidence: 99%

“…Rosetta Pairwise Interaction Score Calculations-Average interaction scores between pairwise interacting residues were computed using Rosetta's per residue energy breakdown protocol as described previously (55). The strength between all possible pairs of interacting amino acid residues within the G protein were calculated across the previously published ensembles of 10 structures after an initial energy minimization using the DualSpace relaxation protocol (56).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

A Conserved Hydrophobic Core in Gαi1 Regulates G Protein Activation and Release from Activated Receptor

Kaya¹,

Lokits

Gilbert³

et al. 2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

G protein-coupled receptor-mediated heterotrimeric G protein activation is a major mode of signal transduction in the cell. Previously, we and other groups reported that the ␣5 helix of G␣ i1 , especially the hydrophobic interactions in this region, plays a key role during nucleotide release and G protein activation. To further investigate the effect of this hydrophobic core, we disrupted it in G␣ i1 by inserting 4 alanine amino acids into the ␣5 helix between residues Gln 333 and Phe 334 (Ins4A). This extends the length of the ␣5 helix without disturbing the ␤6-␣5 loop interactions. This mutant has high basal nucleotide exchange activity yet no receptor-mediated activation of nucleotide exchange. By using structural approaches, we show that this mutant loses critical hydrophobic interactions, leading to significant rearrangements of side chain residues His 57 , Phe 189 , Phe 191 , and Phe 336 ; it also disturbs the rotation of the ␣5 helix and the -interaction between His 57 and Phe 189 . In addition, the insertion mutant abolishes G protein release from the activated receptor after nucleotide binding. Our biochemical and computational data indicate that the interactions between ␣5, ␣1, and ␤2-␤3 are not only vital for GDP release during G protein activation, but they are also necessary for proper GTP binding (or GDP rebinding). Thus, our studies suggest that this hydrophobic interface is critical for accurate rearrangement of the ␣5 helix for G protein release from the receptor after GTP binding.Heterotrimeric G proteins, composed of ␣, ␤, and ␥ subunits, act as a molecular switches that turn on intracellular signaling cascades in response to the activation of G protein-coupled receptors by extracellular stimuli. Therefore, G proteins have a critical role in many different cellular responses (1-6).The G␣ subunit binds GDP and forms a tight complex with the G␤␥ subunits. Activated G protein-coupled receptors can catalyze the exchange of GDP for GTP, which leads to the dissociation of the receptor-G protein complex into isolated receptor and G␣ and G␤␥ subunits. Both the G␣ and G␤␥ subunits can then stimulate or inhibit downstream effectors. Signal propagation ceases after the G␣ subunit hydrolyzes GTP, returns to the inactive state, and rebinds to the G␤␥ subunit, regenerating the GDP-bound heterotrimeric state.Previous studies showed that the activated receptor directly interacts with the G protein by binding to the C-terminal ␣5 helix of G␣, inducing a rigid body rotation and translation that pull this helix into a hydrophobic pocket on the receptor (7,8). This leads to the rearrangement of the interfaces between helices ␣5, ␣1, and the ␤2-␤3 strands and between ␣5 and the ␤6-␣5 loop (1, 7, 9 -11). Residue Phe 336 in the ␣5 helix is highly conserved in small (12, 13) and large GTPases (14) in both the animal and plant kingdoms (15-18). Our in silico results predicted that Phe 336 is the most energetically important residue both in maintaining the basal state and in promoting the receptor-bound conformation (6...

show abstract

Section: Methodsmentioning

confidence: 99%