Selectivity determinants of GPCR-G-protein binding.
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ABSTRACT:
SUBMITTER: Flock T
PROVIDER: S-EPMC5846738 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:G protein-coupled receptors (GPCRs) activate four families of heterotrimeric G proteins, and individual receptors must select a subset of G proteins to produce appropriate cellular responses. Although the precise mechanisms of coupling selectivity are uncertain, the Gα subunit C terminus is widely believed to be the primary determinant recognized by cognate receptors. Here, we directly assess coupling between 14 representative GPCRs and 16 Gα subunits, including one wild-type Gα subunit from each of the four families and 12 chimeras with exchanged C termini. We use a sensitive bioluminescence resonance energy transfer (BRET) assay that provides control over both ligand and nucleotide binding, and allows direct comparison across G protein families. We find that the Gs- and Gq-coupled receptors we studied are relatively promiscuous and always couple to some extent to Gi1 heterotrimers. In contrast, Gi-coupled receptors are more selective. Our results with Gα subunit chimeras show that the Gα C terminus is important for coupling selectivity, but no more so than the Gα subunit core. The relative importance of the Gα subunit core and C terminus is highly variable and, for some receptors, the Gα core is more important for selective coupling than the C terminus. Our results suggest general rules for GPCR-G protein coupling and demonstrate that the critical G protein determinants of selectivity vary widely, even for different receptors that couple to the same G protein.
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Project description:G protein-coupled receptors (GPCRs) regulate diverse physiological events, which makes them as the major targets for many approved drugs. G proteins are downstream molecules that receive signals from GPCRs and trigger cell responses. The GPCR-G protein selectivity mechanism on how they properly and timely interact is still unclear. Here, we analyzed model GPCRs (i.e. HTR, DAR) and Gα proteins with a coevolutionary tool, statistical coupling analysis. The results suggested that 5-hydroxytryptamine receptors and dopamine receptors have common conserved and coevolved residues. The Gα protein also have conserved and coevolved residues. These coevolved residues were implicated in the molecular functions of the analyzed proteins. We also found specific coevolving pairs related to the selectivity between GPCR and G protein were identified. We propose that these results would contribute to better understandings of not only the functional residues of GPCRs and Gα proteins but also GPCR-G protein selectivity mechanisms.
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Project description:Malic enzymes have high cofactor selectivity. An isoform-specific distribution of residues 314, 346, 347 and 362 implies that they may play key roles in determining the cofactor specificity. Currently, Glu314, Ser346, Lys347 and Lys362 in human c-NADP-ME were changed to the corresponding residues of human m-NAD(P)-ME (Glu, Lys, Tyr and Gln, respectively) or Ascaris suum m-NAD-ME (Ala, Ile, Asp and His, respectively). Kinetic data demonstrated that the S346K/K347Y/K362Q c-NADP-ME was transformed into a debilitated NAD?-utilizing enzyme, as shown by a severe decrease in catalytic efficiency using NADP? as the cofactor without a significant increase in catalysis using NAD? as the cofactor. However, the S346K/K347Y/K362H enzyme displayed an enhanced value for k(cat,NAD), suggesting that His at residue 362 may be more beneficial than Gln for NAD? binding. Furthermore, the S346I/K347D/K362H mutant had a very large K(m,NADP) value compared to other mutants, suggesting that this mutant exclusively utilizes NAD? as its cofactor. Since the S346K/K347Y/K362Q, S346K/K347Y/K362H and S346I/K347D/K362H c-NADP-ME mutants did not show significant reductions in their K(m,NAD) values, the E314A mutation was then introduced into these triple mutants. Comparison of the kinetic parameters of each triple-quadruple mutant pair (for example, S346K/K347Y/K362Q versus E314A/S346K/K347Y/K362Q) revealed that all of the K(m) values for NAD? and NADP(+) of the quadruple mutants were significantly decreased, while either k(cat,NAD) or k(cat,NADP) was substantially increased. By adding the E314A mutation to these triple mutant enzymes, the E314A/S346K/K347Y/K362Q, E314A/S346K/K347Y/K362H and E314A/S346I/K347D/K362H c-NADP-ME variants are no longer debilitated but become mainly NAD?-utilizing enzymes by a considerable increase in catalysis using NAD? as the cofactor. These results suggest that abolishing the repulsive effect of Glu314 in these quadruple mutants increases the binding affinity of NAD?. Here, we demonstrate that a series of E314A-containing c-NADP-ME quadruple mutants have been changed to NAD?-utilizing enzymes by abrogating NADP? binding and increasing NAD? binding.
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Project description:IQ motif-containing GTPase-activating proteins (IQGAPs) modulate a wide range of cellular processes by acting as scaffolds and driving protein components into distinct signaling networks. Their functional states have been proposed to be controlled by members of the RHO family of GTPases, among other regulators. In this study, we show that IQGAP1 and IQGAP2 can associate with CDC42 and RAC1-like proteins but not with RIF, RHOD, or RHO-like proteins, including RHOA. This seems to be based on the distribution of charged surface residues, which varies significantly among RHO GTPases despite their high sequence homology. Although effector proteins bind first to the highly flexible switch regions of RHO GTPases, additional contacts outside are required for effector activation. Sequence alignment and structural, mutational, and competitive biochemical analyses revealed that RHO GTPases possess paralog-specific residues outside the two highly conserved switch regions that essentially determine the selectivity of RHO GTPase binding to IQGAPs. Amino acid substitution of these specific residues in RHOA to the corresponding residues in RAC1 resulted in RHOA association with IQGAP1. Thus, electrostatics most likely plays a decisive role in these interactions.
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Project description:BackgroundMembers of the periplasmic binding protein (PBP) superfamily utilize a highly conserved inter-domain ligand binding site that adapts to specifically bind a chemically diverse range of ligands. This paradigm of PBP ligand binding specificity was recently altered when the structure of the Thermotoga maritima cellobiose-binding protein (tmCBP) was solved. The tmCBP binding site is bipartite, comprising a canonical solvent-excluded region (subsite one), adjacent to a solvent-filled cavity (subsite two) where specific and semi-specific ligand recognition occur, respectively.ResultsA molecular level understanding of binding pocket adaptation mechanisms that simultaneously allow both ligand specificity at subsite one and promiscuity at subsite two has potentially important implications in ligand binding and drug design studies. We sought to investigate the determinants of ligand binding selectivity in tmCBP through biophysical characterization of tmCBP in the presence of varying β-glucan oligosaccharides. Crystal structures show that whilst the amino acids that comprise both the tmCBP subsite one and subsite two binding sites remain fixed in conformation regardless of which ligands are present, the rich hydrogen bonding potential of water molecules may facilitate the ordering and the plasticity of this unique PBP binding site.ConclusionsThe identification of the roles these water molecules play in ligand recognition suggests potential mechanisms that can be utilized to adapt a single ligand binding site to recognize multiple distinct ligands.
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Project description:Nickel is an essential micronutrient for the survival of many microbes. On account of the toxicity of nickel and its scarcity in the environment, microbes have evolved specific systems for uptaking and delivering nickel to enzymes. NikA, the solute binding protein for the ATP-binding cassette (ABC) importer NikABCDE, plays a vital role in the nickel homeostasis of Escherichia coli by selectively binding nickel over other metals in the metabolically complex periplasm. While the endogenous ligand for NikA is known to be the Ni(II)-(L-His)2 complex, the molecular basis by which NikA selectively binds Ni(II)-(L-His)2 is unclear, especially considering that NikA can bind multiple metal-based ligands with comparable affinity. Here we show that, regardless of its promiscuous binding activity, NikA preferentially interacts with Ni(II)-(L-His)2, even over other metal-amino acid ligands with an identical coordination geometry for the metal. Replacing both the Ni(II) and the L-His residues in Ni(II)-(L-His)2 compromises binding of the ligand to NikA, in part because these alterations affect the degree by which NikA closes around the ligand. Replacing H416, the only NikA residue that ligates the Ni(II), with other potential metal-coordinating amino acids decreases the binding affinity of NikA for Ni(II)-(L-His)2 and compromises uptake of Ni(II) into E. coli cells, likely due to altered metal selectivity of the NikA mutants. Together, the biochemical and in vivo studies presented here define key aspects of how NikA selects for Ni(II)-(L-His)2 over other metal complexes, and can be used as a reference for studies into the metal selectivity of other microbial solute binding proteins.
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Project description:This SuperSeries is composed of the SubSeries listed below.
2024-06-01 | GSE252435 | GEO