- Email: [email protected]
An Acid Test for G Proteins
Molecular Cell
Previews An Acid Test for G Proteins Stephen R. Sprang1,* 1Center for Biomolecular Structure and Dynamics, Division of Biology, The University of Montana, Missoula, MT 59812, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.08.012
In this issue, Isom et al. (2013) report their exciting discovery that G proteins can sense pH changes to finetune signaling in response to metabolic changes. Heterotrimeric G proteins are cytoplasmic transducers of signals generated by ligand-activated 7-transmembrane receptors (G protein-coupled receptors, or GPCRs) embedded in the plasma membranes of cells. GPCRs activate G proteins by catalyzing exchange of GDP for GTP at the a subunit (Ga) with the concomitant release of the Gbg heterodimer. Separately or together, Ga,GTP and Gbg stimulate enzymes that regulate ion channels, Ras-family G proteins, and enzymes that produce second messengers (e.g., cyclic AMP) (Cabrera-Vera et al., 2003). In yeast, GPCRs intercept mating pheromones and monitor extracellular glucose concentration and so generate intracellular signals either to arrest growth or to proliferate. In this issue, Isom et al. show that Gpa1, the Ga subunit involved in the mating pheromone response, is also regulated by intracellular pH, reaffirming a growing recognition that protons, like other mono- and divalent cations, have roles as cellular second messengers (Isom et al., 2013). The path to this discovery began with the structural problem, how proteins can accommodate buried ionizable amino acid side chains in their interiors, and now arrives at a better understanding of the mechanisms by which regulatory proteins evolve properties as transducers of proton-mediated signaling. Key to the proton-sensing properties of a protein is the distribution of its ionizable amino acid side chains. With the exception of histidine, the pKas of acidic and basic amino acid side chains are far from the normal range of intracellular pH (centered at about 7.2). Within that range in aqueous solution they are charged and form strong polar interactions with solvent water molecules. Before the structure of the first protein had been determined, Kauzmann
correctly deduced that, in the folded tertiary structures of proteins, the great majority of ionizable side chains would be located at the protein surface (Kauzmann, 1959). However, to function as catalysts or transporters, many proteins must harbor buried and potentially charged side chains. Internalization of a normally charged side chain destabilizes the native fold of the protein. There is an energetic penalty for stripping solvent water molecules from charged side chains, and then either burying the charges in the hydrophobic protein interior, or in shifting the pKa of the side chain to near-neutral pH. Earlier, Isom, Garcia-Moreno, and colleagues had shown that lysine and glutamate residues introduced into the interior of a mutationally stabilized staphylococcal nuclease have pKas shifted by several units toward neutral pH (Isom et al., 2008). Such buried side chains therefore titrate near-neutral, physiological pH. Protonation or deprotonation would introduce a charge into the protein interior. The ideal proton sensor would be capable of converting the energetic cost of charge burial into useful work, for example, by undergoing a conformational change that both solvates the charged moieties (reducing the energy of the system) while altering the functional properties of the protein. Burial of ionizable or—even better—clusters of ionizable residues at protein domain interfaces could afford a mechanism to induce such functionally transformative conformational changes. To discover potential proton sensors, Isom and colleagues canvassed the protein databank using an algorithm to identify proteins with clusters of buried ionizable residues. About 10% do, and Ga subunits are among them (Figure 1). Members of the superfamily of Ras GTPases, Ga are distinguished from other
Ras-like proteins by a helical domain inserted near a mobile polypeptide segment that is involved in binding and hydrolysis of GTP. While the helical domain does not interact tightly with the nucleotide, it does help to shield it from solvent. Indeed, the helical and Ras domains of Ga rotate 120 apart when the heterotrimer binds to an activated GPCR, an event that accompanies nucleotide release from Ga (Rasmussen et al., 2011). Even when not bound to a receptor, Ga might fluctuate between domainopen and domain-closed states, perhaps dynamically modulating its ability to interact with its effectors or with Gbg. Isom et al. find that various physical properties of Gpa1 (as well as Gai1, its mammalian homolog) change rapidly in the pH 6–7 range: thermal stability decreases, while accessibility of two partly occluded cysteine residues increases. Two-dimensional NMR spectra show that some Ga residues occupy chemically distinct environments at pH 6 and 8, while populations of both are observed at neutrality. These physical changes likely accompany titration and subsequent solvation of residues that constitute charged cluster. Domain separation (Figure 1) would provide one mechanism (among perhaps others) to both solvate these charges and change the signaling state of Ga. Indeed, the signaling properties of Gpa1 are pH sensitive. Stimulation of the mating factor receptor leads to the phosphorylation and activation of MAP kinases that induce cell-cycle arrest (Dohlman and Slessareva, 2006). In this pathway Gbg is the signal transducer that promotes MAPK activation, and Gpa1,GDP acts as an inhibitor by sequestering Gbg in a heterotrimeric complex. Using metabolic, chemical, and gene-regulatory methods to manipulate intracellular pH,
Molecular Cell 51, August 22, 2013 ª2013 Elsevier Inc. 405
Molecular Cell
Previews
in conjunction with a sensitive erty of G proteins, such that in vivo fluorescent-reporter different members of the protein, Isom et al. show that family are tuned to alter sigMAP kinase phosphorylation naling response within spefollows a parabolic response, cific pH regimes. The findings with a maximum near of Isom et al. have revealed an 6.7 about 1/2 unit below that important target of proton for aerobically metabolizing regulation, offering intriguing yeast. It is worth pointing out avenues for exploration. that glucose restriction drives down intracellular pH (Orij ACKNOWLEDGMENTS et al., 2011). Low pH presumably impedes binding of Gbg Supported in part by NIH grant 1R01GM105993-01 to S.R.S. by Gpa1,GDP, as might be the consequence of a major conformational transition in REFERENCES the G protein. Accordingly, a genetic knockout of the Cabrera-Vera, T.M., Vanhauwe, J., Thomas, T.O., Medkova, M., PreiGTPase-activating protein ninger, A., Mazzoni, M.R., and that promotes formation of Hamm, H.E. (2003). Endocr. Rev. 24, 765–781. Gpa1,GDP also prolongs MAPK activation. Finally, low Dohlman, H.G., and Slessareva, J.E. intracellular pH promotes Figure 1. How a Shift in pH Could Induce a Conformational (2006). Sci. STKE 2006, cm6. Transition in Ga phosphorylation of Gpa1, (A–D) A cluster of ionizable acidic (red) and basic (blue) and two cysteine resIsom, D.G., Cannon, B.R., Castawhich hastens ubiquitination idues (yellow) in Ga1 bound to a GTP analog (A) have pKas shifted toward n˜eda, C.A., Robinson, A., and and degradation of the G proneutrality and are not exposed on the solvent-accessible surface of the moleGarcı´a-Moreno, B. (2008). Proc. cule (B). Titration of these residues by a change in pH could induce a transition Natl. Acad. Sci. USA 105, 17784– tein (Torres et al., 2011). Other similar to that observed in the b2-adrenergic receptor-bound complex of Gs 17788. substrates of the kinase are (Rasmussen et al., 2011) (C), in which the Ras (R) and helical (H) domains not affected, showing that are separated and ionizable residues are exposed and solvated (D). This is a Isom, D.G., Sridharan, V., Baker, R., hypothetical transition, presented only to illustrate a possible mechanism of Clement, S.T., Smalley, D.M., and increased susceptibility to Dohlman, H.G. (2013). Mol. Cell 51, pH sensing. phosphorylation at low pH is this issue, 531–538. a property of Gpa1 itself. Because the network of buried ioniz- glucose to starved yeast generates a Kauzmann, W. (1959). Adv. Protein Chem. 14, able residues present in Gpa1 is widely burst of glycolysis that further reduces 1–63. conserved among the nearly 200 intracellular pH, followed a return to Orij, R., Brul, S., and Smits, G.J. (2011). Biochim. sequenced a subunits, it is likely that neutral pH as ATP-driven proton pumps Biophys. Acta 1810, 933–944. many of these Ga proteins are proton sen- become active. We can speculate that Rasmussen, S.G., DeVree, B.T., Zou, Y., Kruse, sors, as well. In yeast, Gpa2 stimulates Gpa2 activity and consequent PKA A.C., Chung, K.Y., Kobilka, T.S., Thian, F.S., Chae, P.S., Pardon, E., Calinski, D., et al. (2011). adenylyl cyclase upon activation by a pathway activation might also be in flux Nature 477, 549–555. glucose-sensing GPCR, thereby upregu- during these rapid changes in intracellular Torres, M.P., Clement, S.T., Cappell, S.D., and lating cAMP-activated protein kinase pH. It will be of interest to learn to what Dohlman, H.G. (2011). J. Biol. Chem. 286, 20208– (PKA) signaling pathways. Restoration of extent pH sensing is an evolvable prop- 20216.
406 Molecular Cell 51, August 22, 2013 ª2013 Elsevier Inc.
Previews An Acid Test for G Proteins Stephen R. Sprang1,* 1Center for Biomolecular Structure and Dynamics, Division of Biology, The University of Montana, Missoula, MT 59812, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2013.08.012
In this issue, Isom et al. (2013) report their exciting discovery that G proteins can sense pH changes to finetune signaling in response to metabolic changes. Heterotrimeric G proteins are cytoplasmic transducers of signals generated by ligand-activated 7-transmembrane receptors (G protein-coupled receptors, or GPCRs) embedded in the plasma membranes of cells. GPCRs activate G proteins by catalyzing exchange of GDP for GTP at the a subunit (Ga) with the concomitant release of the Gbg heterodimer. Separately or together, Ga,GTP and Gbg stimulate enzymes that regulate ion channels, Ras-family G proteins, and enzymes that produce second messengers (e.g., cyclic AMP) (Cabrera-Vera et al., 2003). In yeast, GPCRs intercept mating pheromones and monitor extracellular glucose concentration and so generate intracellular signals either to arrest growth or to proliferate. In this issue, Isom et al. show that Gpa1, the Ga subunit involved in the mating pheromone response, is also regulated by intracellular pH, reaffirming a growing recognition that protons, like other mono- and divalent cations, have roles as cellular second messengers (Isom et al., 2013). The path to this discovery began with the structural problem, how proteins can accommodate buried ionizable amino acid side chains in their interiors, and now arrives at a better understanding of the mechanisms by which regulatory proteins evolve properties as transducers of proton-mediated signaling. Key to the proton-sensing properties of a protein is the distribution of its ionizable amino acid side chains. With the exception of histidine, the pKas of acidic and basic amino acid side chains are far from the normal range of intracellular pH (centered at about 7.2). Within that range in aqueous solution they are charged and form strong polar interactions with solvent water molecules. Before the structure of the first protein had been determined, Kauzmann
correctly deduced that, in the folded tertiary structures of proteins, the great majority of ionizable side chains would be located at the protein surface (Kauzmann, 1959). However, to function as catalysts or transporters, many proteins must harbor buried and potentially charged side chains. Internalization of a normally charged side chain destabilizes the native fold of the protein. There is an energetic penalty for stripping solvent water molecules from charged side chains, and then either burying the charges in the hydrophobic protein interior, or in shifting the pKa of the side chain to near-neutral pH. Earlier, Isom, Garcia-Moreno, and colleagues had shown that lysine and glutamate residues introduced into the interior of a mutationally stabilized staphylococcal nuclease have pKas shifted by several units toward neutral pH (Isom et al., 2008). Such buried side chains therefore titrate near-neutral, physiological pH. Protonation or deprotonation would introduce a charge into the protein interior. The ideal proton sensor would be capable of converting the energetic cost of charge burial into useful work, for example, by undergoing a conformational change that both solvates the charged moieties (reducing the energy of the system) while altering the functional properties of the protein. Burial of ionizable or—even better—clusters of ionizable residues at protein domain interfaces could afford a mechanism to induce such functionally transformative conformational changes. To discover potential proton sensors, Isom and colleagues canvassed the protein databank using an algorithm to identify proteins with clusters of buried ionizable residues. About 10% do, and Ga subunits are among them (Figure 1). Members of the superfamily of Ras GTPases, Ga are distinguished from other
Ras-like proteins by a helical domain inserted near a mobile polypeptide segment that is involved in binding and hydrolysis of GTP. While the helical domain does not interact tightly with the nucleotide, it does help to shield it from solvent. Indeed, the helical and Ras domains of Ga rotate 120 apart when the heterotrimer binds to an activated GPCR, an event that accompanies nucleotide release from Ga (Rasmussen et al., 2011). Even when not bound to a receptor, Ga might fluctuate between domainopen and domain-closed states, perhaps dynamically modulating its ability to interact with its effectors or with Gbg. Isom et al. find that various physical properties of Gpa1 (as well as Gai1, its mammalian homolog) change rapidly in the pH 6–7 range: thermal stability decreases, while accessibility of two partly occluded cysteine residues increases. Two-dimensional NMR spectra show that some Ga residues occupy chemically distinct environments at pH 6 and 8, while populations of both are observed at neutrality. These physical changes likely accompany titration and subsequent solvation of residues that constitute charged cluster. Domain separation (Figure 1) would provide one mechanism (among perhaps others) to both solvate these charges and change the signaling state of Ga. Indeed, the signaling properties of Gpa1 are pH sensitive. Stimulation of the mating factor receptor leads to the phosphorylation and activation of MAP kinases that induce cell-cycle arrest (Dohlman and Slessareva, 2006). In this pathway Gbg is the signal transducer that promotes MAPK activation, and Gpa1,GDP acts as an inhibitor by sequestering Gbg in a heterotrimeric complex. Using metabolic, chemical, and gene-regulatory methods to manipulate intracellular pH,
Molecular Cell 51, August 22, 2013 ª2013 Elsevier Inc. 405
Molecular Cell
Previews
in conjunction with a sensitive erty of G proteins, such that in vivo fluorescent-reporter different members of the protein, Isom et al. show that family are tuned to alter sigMAP kinase phosphorylation naling response within spefollows a parabolic response, cific pH regimes. The findings with a maximum near of Isom et al. have revealed an 6.7 about 1/2 unit below that important target of proton for aerobically metabolizing regulation, offering intriguing yeast. It is worth pointing out avenues for exploration. that glucose restriction drives down intracellular pH (Orij ACKNOWLEDGMENTS et al., 2011). Low pH presumably impedes binding of Gbg Supported in part by NIH grant 1R01GM105993-01 to S.R.S. by Gpa1,GDP, as might be the consequence of a major conformational transition in REFERENCES the G protein. Accordingly, a genetic knockout of the Cabrera-Vera, T.M., Vanhauwe, J., Thomas, T.O., Medkova, M., PreiGTPase-activating protein ninger, A., Mazzoni, M.R., and that promotes formation of Hamm, H.E. (2003). Endocr. Rev. 24, 765–781. Gpa1,GDP also prolongs MAPK activation. Finally, low Dohlman, H.G., and Slessareva, J.E. intracellular pH promotes Figure 1. How a Shift in pH Could Induce a Conformational (2006). Sci. STKE 2006, cm6. Transition in Ga phosphorylation of Gpa1, (A–D) A cluster of ionizable acidic (red) and basic (blue) and two cysteine resIsom, D.G., Cannon, B.R., Castawhich hastens ubiquitination idues (yellow) in Ga1 bound to a GTP analog (A) have pKas shifted toward n˜eda, C.A., Robinson, A., and and degradation of the G proneutrality and are not exposed on the solvent-accessible surface of the moleGarcı´a-Moreno, B. (2008). Proc. cule (B). Titration of these residues by a change in pH could induce a transition Natl. Acad. Sci. USA 105, 17784– tein (Torres et al., 2011). Other similar to that observed in the b2-adrenergic receptor-bound complex of Gs 17788. substrates of the kinase are (Rasmussen et al., 2011) (C), in which the Ras (R) and helical (H) domains not affected, showing that are separated and ionizable residues are exposed and solvated (D). This is a Isom, D.G., Sridharan, V., Baker, R., hypothetical transition, presented only to illustrate a possible mechanism of Clement, S.T., Smalley, D.M., and increased susceptibility to Dohlman, H.G. (2013). Mol. Cell 51, pH sensing. phosphorylation at low pH is this issue, 531–538. a property of Gpa1 itself. Because the network of buried ioniz- glucose to starved yeast generates a Kauzmann, W. (1959). Adv. Protein Chem. 14, able residues present in Gpa1 is widely burst of glycolysis that further reduces 1–63. conserved among the nearly 200 intracellular pH, followed a return to Orij, R., Brul, S., and Smits, G.J. (2011). Biochim. sequenced a subunits, it is likely that neutral pH as ATP-driven proton pumps Biophys. Acta 1810, 933–944. many of these Ga proteins are proton sen- become active. We can speculate that Rasmussen, S.G., DeVree, B.T., Zou, Y., Kruse, sors, as well. In yeast, Gpa2 stimulates Gpa2 activity and consequent PKA A.C., Chung, K.Y., Kobilka, T.S., Thian, F.S., Chae, P.S., Pardon, E., Calinski, D., et al. (2011). adenylyl cyclase upon activation by a pathway activation might also be in flux Nature 477, 549–555. glucose-sensing GPCR, thereby upregu- during these rapid changes in intracellular Torres, M.P., Clement, S.T., Cappell, S.D., and lating cAMP-activated protein kinase pH. It will be of interest to learn to what Dohlman, H.G. (2011). J. Biol. Chem. 286, 20208– (PKA) signaling pathways. Restoration of extent pH sensing is an evolvable prop- 20216.
406 Molecular Cell 51, August 22, 2013 ª2013 Elsevier Inc.