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Selectivity determinants of GPCR-G-protein binding.

ABSTRACT: The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of amino acids) on each of the 16 human G proteins that is recognized by distinct regions on the approximately 800 human receptors. Although universally conserved positions in the barcode allow the receptors to bind and activate G proteins in a similar manner, different receptors recognize the unique positions of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation for understanding the molecular basis of coupling selectivity within individual receptors and G proteins.

SUBMITTER: Flock T

PROVIDER: S-EPMC5846738 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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Selectivity determinants of GPCR-G-protein binding.

Flock Tilman T Hauser Alexander S AS Lund Nadia N Gloriam David E DE Balaji Santhanam S Babu M Madan MM

Nature 20170510 7654

The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is critical to trigger the appropriate physiological response. However, the determinants of selective binding have remained elusive. Here we reveal the existence of a selectivity barcode (that is, patterns of amino acids) on each of the 16 human G proteins that is recognized by distinct regions on the approximately 800 human receptors. Although universally conserved positions in the barcode allow the receptors t ...[more]

PMID: 28489817

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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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Selectivity determinants of GPCR-G-protein binding.

Publications

Selectivity determinants of GPCR-G-protein binding.

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