Previously, we published an article providing an overview of the Rosetta suite of biomacromolecular modeling software and a series of step-by-step tutorials [Kaufmann, K. W., et al. (2010) Biochemistry 49, 2987–2998]. The overwhelming positive response to this publication we received motivates us to here share the next iteration of these tutorials that feature de novo folding, comparative modeling, loop construction, protein docking, small molecule docking, and protein design. This updated and expanded set of tutorials is needed, as since 2010 Rosetta has been fully redesigned into an object-oriented protein modeling program Rosetta3. Notable improvements include a substantially improved energy function, an XML-like language termed “RosettaScripts” for flexibly specifying modeling task, new analysis tools, the addition of the TopologyBroker to control conformational sampling, and support for multiple templates in comparative modeling. Rosetta’s ability to model systems with symmetric proteins, membrane proteins, noncanonical amino acids, and RNA has also been greatly expanded and improved.

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-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...

BackgroundHeterotrimeric G proteins are fundamental signaling proteins composed of three subunits, Gα and a Gβγ dimer. The role of Gα as a molecular switch is critical for transmitting and amplifying intracellular signaling cascades initiated by an activated G protein Coupled Receptor (GPCR). Despite their biochemical and therapeutic importance, the study of G protein evolution has been limited to the scope of a few model organisms. Furthermore, of the five primary Gα subfamilies, the underlying gene structure of only two families has been thoroughly investigated outside of Mammalia evolution. Therefore our understanding of Gα emergence and evolution across phylogeny remains incomplete.ResultsWe have computationally identified the presence and absence of every Gα gene (GNA-) across all major branches of Deuterostomia and evaluated the conservation of the underlying exon-intron structures across these phylogenetic groups. We provide evidence of mutually exclusive exon inclusion through alternative splicing in specific lineages. Variations of splice site conservation and isoforms were found for several paralogs which coincide with conserved, putative motifs of DNA-/RNA-binding proteins. In addition to our curated gene annotations, within Primates, we identified 15 retrotranspositions, many of which have undergone pseudogenization. Most importantly, we find numerous deviations from previous findings regarding the presence and absence of individual GNA- genes, nuanced differences in phyla-specific gene copy numbers, novel paralog duplications and subsequent intron gain and loss events.ConclusionsOur curated annotations allow us to draw more accurate inferences regarding the emergence of all Gα family members across Metazoa and to present a new, updated theory of Gα evolution. Leveraging this, our results are critical for gaining new insights into the co-evolution of the Gα subunit and its many protein binding partners, especially therapeutically relevant G protein – GPCR signaling pathways which radiated in Vertebrata evolution.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1147-8) contains supplementary material, which is available to authorized users.

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