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Conformational Transitions and the Activation of Heterotrimeric G Proteins by G Protein-Coupled Receptors Christopher Draper-Joyce*,# and Sebastian George Barton Furness*,#
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Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia ABSTRACT: G protein-coupled receptors (GPCRs) are particularly attractive targets for therapeutic pharmaceuticals. This is because they are involved in almost all facets of physiology, in many pathophysiological processes, they are tractable due to their cell surface location, and can exhibit highly textured pharmacology. While the development of new drugs does not require the molecular details of the mechanism of activity for a particular target, there has been increasing interest in the GPCR field in these details. In part, this has come with the recognition that differential activity at a particular target might be a way in which to leverage drug activity, either through manipulation of efficacy or through differential coupling (signaling bias). To this end, the past few years have seen a number of publications that have specifically attempted to address one or more aspects of the molecular reaction pathway, leading to activation of heterotrimeric G proteins by GPCRs. allosteric interactions involved (see below). In this figure, it is also implicit that agonist binding and G protein binding are allosterically coupled. In a live cell environment, this is not easily measured, but in isolated membranes, where one can control the amount of nucleotide in the system, this relationship is very well established with the G protein increasing agonist affinity between 10- and 1000-fold.4 There are now structures for at least six different GPCRs where both an antagonist bound receptor and agonist bound ternary complex have been solved (reviewed in ref 5). Common to all these pairs is that there are large differences in the receptor conformation, particularly at the intracellular face where G protein binding occurs and also through the central transmembrane bundle to the ligand-binding pocket, where there are numerous side-chain rearrangements along with differences in the relative orientations of the transmembrane helices. In counterpoint to this, there are now receptor-bound conformations for the Gαβγ heterotrimer along with GDP bound, and, in some cases, GTP bound equivalents (e.g., refs 6−13). For these structures, the conformational differences between the Gα subunit when bound to either GDP and Gβγ or GTP are relatively small (confined predominantly to switch II rearrangements) compared to the differences that exist when comparing either nucleotide bound state to the receptorbound, nucleotide-free state (e.g., ref 14 and Figure 2). High stability of the ternary, nucleotide-free state is implicit in the ternary complex model, but this stability should necessarily be limited to properly allow acceleration of nucleotide exchange at the G protein. Thus, when considering activation of a GPCR, it is useful to consider this as part of a reaction coordinate pathway, similar to how enzyme kinetics are considered, as previously proposed elsewhere.15,16 In this model, there are distinct steps in the pathway that are depicted in Figure 3; there are several active states that are capable of rapid interconversion, and there is no singular “active state”.
G
protein-coupled receptors (GPCRs) exhibit conformational flexibility on time scales ranging from femtosecond (bond vibration) through to millisecond and second (large conformational rearrangements), as is generally seen for proteins which act as molecular machines.1 Conformational sampling at longer (μs−ms) time scales has been well described for enzyme systems.2 For enzymes, these longtime-scale exchanges are intimately tied to their ability to act as catalysts for their respective reactions and involve relatively large conformational changes separated by comparably highenergy barriers.3 For GPCRs, it is not yet clear how large-scale conformational exchange is related to function, however recent work by a number of groups is starting to hint at some of the important roles this type of conformational sampling has for these receptors.
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A REACTION COORDINATE PATHWAY The role of a GPCR is to act as an agonist-dependent activator of heterotrimeric G proteins (henceforth G proteins) and nonG protein signaling transducers such as β-arrestins. In this viewpoint, we are only going to discuss G proteins. With respect to G proteins, GPCRs act as enzymatic guanine nucleotide exchange factors (GEFs) sponsoring the exchange of GDP from the inactive G protein heterotrimer to a GTP bound Gα and dissociation of this from the Gβγ dimer. To achieve this, there must be a transition from GPCR conformations with low GEF activity to conformations with significantly higher GEF activity. For the G protein, it must transition from GDP bound conformations through nucleotide-free conformations to ones bound to GTP along with dissociation of Gα and Gβγ subunits. This is often represented pictorially, as shown in Figure 1. As shown in this figure, there is a clear direction for this pathway where the relative abundance of intracellular GTP over GDP (approximately 10fold excess) makes the forward direction energetically favorable, and the GPCR is acting by lowering the activation energy. The relative affinity of the G protein for GDP and GTP may also contribute to the forward reaction, however, this contribution is difficult to quantify due to the additional © XXXX American Chemical Society
Received: July 19, 2019
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DOI: 10.1021/acsptsci.9b00054 ACS Pharmacol. Transl. Sci. XXXX, XXX, XXX−XXX
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Figure 3. A reaction coordinate pathway for the activation of heterotrimeric G proteins by GPCRs. Ligand binds receptor (A−B), GDP bound heterotrimer interacts with receptor (B−C/1−2) leading to (D/3) nucleotide release, followed by a conformational change (E/ 4) to enable nucleotide-binding (F/5) and subsequent release of GTP bound GTP Gα and Gβγ (G/6) and ligand unbinding (H). The receptor can cycle back to a state competent to sponsor another round of nucleotide exchange either directly after release of G protein or upon rebinding ligand (shown by arrows above). After interaction with downstream effectors, the G proteins are recycled for another round of activation dependent on GTPase activity (arrows below).
interaction, it is difficult to see how low constitutive activity could be effectively maintained if GPCRs were intrinsically capable of exploring conformational space with high GEF activity (see below). There are several experimental approaches to assess conformational transitions; nuclear magnetic resonance (NMR) experiments with isotopic labeling of native side chains (e.g., 13C methionine),17,18 NMR with chemical side chain labeling such as 13C dimethyl-lysine,19 and 19 20−22 F or nitroxide spin labeling21,23 of cysteine residues and approaches using fluorescent modifications relying either on changes in intrinsic fluorescence24 dependent on local environment or on Fö rster resonance energy transfer (FRET).25 In all cases, any changes in conformations or conformational dynamics are being viewed through the lens of the modification; absence of an induced change in signal is only evidence that the particular residue has not experienced a measurable change in conformational sampling, not that the overall protein has, or has not, undergone conformational changes. Similarly, only a very small selection of GPCRs have been subject to this kind of analysis, and it is not yet clear how widely applicable the observed effects are likely to be. Current NMR data from the β2, A2A, μ-opioid receptors, and
Figure 1. Simplified, textbook view of activation of heterotrimeric G proteins by GPCRs. In the basal state (A), the G protein is in an inactive heterotrimeric GDP bound state. Upon ligand binding to the GPCR, the ligand bound GPCR is able to sponsor nucleotide exchange at the heterotrimeric G protein (B), leading to release of active GTP Gα and Gβγ subunits (C).
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CONFORMATIONAL TRANSITIONS AT THE LEVEL OF THE RECEPTOR Many GPCRs exhibit very low constitutive activity, suggesting that, in the absence of an agonist, these receptors either explore conformational space that has low GEF activity or spend almost no time sampling conformations capable of high GEF activity. Given the allosteric nature of the GPCR-G protein
Figure 2. A comparison of the conformation of Gαs in the GDP (6EG8), nucleotide-free (3SN6), and GTP (GTPγS, 1AZT) bound states. Heatmap of structural conservation between different Gαs states colored according to α-carbon root-mean-square deviation (α-C RMSD) (Å) between different structures (scale on right in Å). (A) Comparison of G0 (white; PDB 3SN6) and GDP bound (colored; PDB 6EG8) Gαs, illustrating the conformational differences in the Ras-like and α-helical domains (see text). (B) Comparison of G0 (white; PDB 3SN6) and GTPγS bound (colored; PDB 1AZT), illustrating the conformational differences in the Ras-like and α-helical domains (see text). (C) Comparison of GDP bound (white; PDB 6EG8) and GTPγS bound (colored; PDB 1AZT), illustrating the high degree of conformational similarity in these states (see text). B
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Figure 4. Alignment of Gαs in the G0 (green; PDB 3SN6) and GDP bound (blue; PDB 6EG8) conformation. Inset depicts side-chain rearrangements of residues within the GDP (purple) binding pocket. Residues in the inset are labeled according to both their native number and the common Gα numbering system (CGN).31
Prosser’s laboratory20 support a model in which agonist binding is entropically driven, with the agonist-only bound receptor exhibiting significantly greater dynamics than either apo or ternary complexes. That the agonist-only bound conformations are not the same as those in the ternary complex provides a mechanism by which conformational selection (for the ternary complex conformation) could provide the necessary binding energy for interaction with the G protein. In the ternary complex of light-activated rhodopsin in nanodiscs, there is still significant conformational sampling by the receptor, with greater overlap with the light-activated transducin-free rhodopsin than is seen in the other ternary receptor complexes, or indeed rhodopsin, in detergent micelles.23 All of these NMR experiments are performed at steady state, thus the mechanism by which the receptor transitions from one set of conformational populations to another is currently unexplored, and there is no data to assess the receptor conformation(s) that exist for a ternary complex in which GDP is still bound. Recent cryo-EM data from Brian Kobilka’s laboratory on the neurotensin receptor coupled to a noncognate G protein suggest that there is at least one additional receptor conformation in the G0 ternary state,28 and hydrogen-deuterium exchange experiments from the same laboratory support the existence of a β2 receptor transition state present after G protein binding and before conversion to conformations consistent with structural data. Careful kinetic evaluation of transducin activation shows that there is a rhodopsin:GαGDPβγ complex that exists prior to GDP release,29 however the conformations of rhodopsin and their relationship to those for the light-activated G protein-free and GGDP state are not known. In single-molecule FRET experiments on the β2 receptor in detergent micelles, reporters located at the intracellular face of the receptor were used to report on ligand- and G protein-induced conformational changes.25 In contrast to NMR experiments, this probe set did not report multiple receptor conformations, either due to conformational exchange faster than acquisition or a limitation in the reporter location. These experiments did examine the receptor conformational change in response to nucleotide binding at a prebound ternary complex and showed that the rate at which the receptor transitioned to a non-G 0
rhodopsin provide some information about conformational transitions that occur in response to ligand (Figure 3, step B), transducer binding (Figure 3 either step D or E), and G protein release (Figure 3, step G to H). With the exception of rhodopsin, the experiments published have replaced the G protein with either a peptide corresponding to the c-terminus of the relevant G protein or a nanobody mimetic and the native plasma membrane replaced with a detergent micelle. It is unclear how closely either nanobodies or the free C-terminal peptide act as mimetics that reflect receptor conformational dynamics in a G protein-bound state. Both C-terminal peptides and G protein mimetic nanobodies provide cooperative binding, as demonstrated by an increase in the high-affinity state of the receptor. In the β2 receptor-bound to both nanobody26 and the heterotrimeric G protein partner,11 there are relatively subtle differences including differences in apparent mobility of the extracellular face of the receptor and a 3 Å difference in the displacement of helix 6. In these NMR studies, the reporters, in all receptors, indicate that the apo state of the receptor samples at least two different conformations. Binding of either agonists or inverse agonists appears to be consistent with conformational selection, including in the case of rhodopsin.27 In the case of agonist binding, the agonist bound state samples one (or more) of the apo states but is also then able to sample states that were so sparsely populated as to be below the level of detection. In cases where a G protein mimetic has been used in the absence of agonist, the G protein mimetic similarly allows the receptor to explore previously invisible states. The co-binding of agonist and G protein mimetic (ternary complex) then causes the receptor to populate a state that was either sparsely populated or invisible, consistent with their reciprocal co-operativity. In some cases, the ternary complex appears to be able to sample several conformational states,19,20,23 while this is not seen with others.17,18,21 The difference in conformational space explored by the receptor in agonist bound, compared with a ternary complex state, is consistent with the published structures where this comparison can be made; in these structural comparisons, the agonist-only bound state has some, but not all the conformational shifts away from the antagonist/apo state that are seen in the ternary complex. Data from Scott C
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Figure 5. Alignment of Gαs in the G0 (green; PDB 3SN6) and GTPγS bound (pink; PDB 1AZT) conformation. Inset depicts side-chain rearrangements of residues within the GTPγS (cyan) binding pocket that contact the γ-phosphate of GTPγS and a subset of side-chain rearrangements in the switch II region that contacts Gβ. Residues in the inset are labeled according to both their native number and the common Gα numbering system (CGN).31
6). In published receptor-G protein complexes, the switch II of the Gα is arranged such that most of the interactions it provides with Gβ are preserved (Figure 5). In addition, the overall relationship between Gα and Gβ in the G0 state are very similar to those in the GDP bound heterotrimer (Figure 4). The pathway of conformational changes that the Gα takes to transition from GDP bound to G0 and from this state to dissociation from Gβγ and GTP binding has not been extensively explored, however recent computational modeling supports the existence of a transition-state intermediate to the known GDP and receptor:G0 states.35 Early data on free Gα suggested that GDP dissociation was the rate-limiting step,36 however in the presence of receptor and Gβγ, a transition step, independent of GDP release, was proposed to be rate limiting.33 The measurement of the conformational change of the β2 receptor ternary complex in response to GDP and GTP was interpreted as GDP release being the rate-limiting step for G protein activation,25 however recent data from the same laboratory suggest that other conformational transitions, including GTP binding, may be rate limiting,37 consistent with our observations for the calcitonin receptor16 and an earlier report on the dopamine D2 receptor.38 For the small G protein, Ras, both release of GDP and binding of GTP are largely driven by conformational selection.39 If this situation also exists for the heterotrimeric G proteins, as might be expected, then there must necessarily be sampling of conformations that are similar to the GTP bound conformations while the G protein is in the receptor-bound G0 state and highlights the conformational sampling through which the G protein must pass in order to be activated.
conformation was faster in the presence of GTP compared with GDP, however the temporal relationship between these conformational changes and G protein dissociation is not known. For rhodopsin, the transition to an inactive conformation occurs after release of all-trans-retinal occurs, and a fusion of the c-terminal tail of transducin to rhodopsin further inhibits the release of all-trans-retinal.27 This raises the possibility that ligand release and G protein release are, in fact, coupled events.
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CONFORMATIONAL TRANSITIONS OF THE G PROTEIN In all published cryo-EM structures, the G protein is present in the G0 state and exhibits a highly dynamic conformation with respect to the Gα α-helical domain that is reflected in poorly resolved density, however the conformation of the Ras-like domain is strikingly similar across the 6 different G proteins in 3 different classes with 13 different receptors (e.g., refs 12, 13, and 30), consistent with a common allosteric activation mechanism.31 Compared with either GDP or GTP bound states, the G0 structure of the Ras-like domain exhibits an approximate 5 Å translation and 60° rotation of the α5 helix that interacts with the receptor (Figures 2, 4, and 5). This difference in the α5 helix is coupled to changes in the loop connecting the bottom of this helix with the β6 strand, a region which otherwise contacts the purine ring of GDP/GTP. This change alters interactions of this region with the α1 helix along with the P-loop that is responsible for contacting the βphosphate of GDP (Figure 4). This is generally accepted as a mechanism for allosteric coupling between the receptor and GDP. Indeed in the absence of receptor, the dissociation rate of GDP from the Gαβγ complex is in the order of 0.015 min−1(at 30 °C).32,33 The Gβγ dimer acts as a guanine nucleotide dissociation inhibitor (GDI) for GDP, increasing the affinity Gα has for GDP by more than 100-fold.34 The GTP-dependent dissociation of Gβγ from Gα depends on the reorganization of the switch II region (and consequently switch I) of Gα, which provides a mutually exclusive interaction interface with Gβ and the γ-phosphate of GTP (Figure 5 and
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IMPLICATIONS OF THE MODEL The reaction coordinate or kinetic model for G protein activation has a number of implications that may help to understand existing data. First, if there are a number of interconverting active states of the receptor:Gαβγ complex, then a change in rate at any one of these steps would provide an elegant mechanism for partial agonism and would be consistent with data on rates of transition.16,25 Thus, partial D
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(8) Noel, J. P., Hamm, H. E., and Sigler, P. B. (1993) The 2.2 A crystal structure of transducin-alpha complexed with GTP gamma S. Nature 366, 654−663. (9) Coleman, D. E., Berghuis, A. M., Lee, E., Linder, M. E., Gilman, A. G., and Sprang, S. R. (1994) Structures of active conformations of Gi alpha 1 and the mechanism of GTP hydrolysis. Science 265, 1405− 1412. (10) Sunahara, R. K., Tesmer, J. J., Gilman, A. G., and Sprang, S. R. (1997) Crystal structure of the adenylyl cyclase activator Gsalpha. Science 278, 1943−1947. (11) Rasmussen, S. G. F., DeVree, B. T., Zou, Y., Kruse, A. C., Chung, K. Y., Kobilka, T. S., Thian, F. S., Chae, P. S., Pardon, E., Calinski, D., Mathiesen, J. M., Shah, S. T. A., Lyons, J. A., Caffrey, M., Gellman, S. H., Steyaert, J., Skiniotis, G., Weis, W. I., Sunahara, R. K., and Kobilka, B. K. (2011) Crystal structure of the β2 adrenergic receptor-Gs protein complex. Nature 477, 549−555. (12) Liang, Y.-L., Khoshouei, M., Radjainia, M., Zhang, Y., Glukhova, A., Tarrasch, J., Thal, D. M., Furness, S. G. B., Christopoulos, G., Coudrat, T., Danev, R., Baumeister, W., Miller, L. J., Christopoulos, A., Kobilka, B. K., Wootten, D., Skiniotis, G., and Sexton, P. M. (2017) Phase-plate cryo-EM structure of a class B GPCR-G-protein complex. Nature 546, 118−123. (13) Draper-Joyce, C. J., Khoshouei, M., Thal, D. M., Liang, Y.-L., Nguyen, A. T. N., Furness, S. G. B., Venugopal, H., Baltos, J.-A., Plitzko, J. X. R. M., Danev, R., Baumeister, W., May, L. T., Wootten, D., Sexton, P. M., Glukhova, A., and Christopoulos, A. (2018) Structure of the adenosine bound human adenosine A. Nature 558, 559−563. (14) Mixon, M. B., Lee, E., Coleman, D. E., Berghuis, A. M., Gilman, A. G., and Sprang, S. R. (1995) Tertiary and quaternary structural changes in Gi alpha 1 induced by GTP hydrolysis. Science 270, 954− 960. (15) Roberts, D. J., and Waelbroeck, M. (2004) G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction? Biochem. Pharmacol. 68, 799−806. (16) Furness, S. G. B., Liang, Y.-L., Nowell, C. J., Halls, M. L., Wookey, P. J., Dal Maso, E., Inoue, A., Christopoulos, A., Wootten, D., and Sexton, P. M. (2016) Ligand-Dependent Modulation of G Protein Conformation Alters Drug Efficacy. Cell 167, 739−749. (17) Kofuku, Y., Ueda, T., Okude, J., Shiraishi, Y., Kondo, K., Maeda, M., Tsujishita, H., and Shimada, I. (2012) Efficacy of the β2adrenergic receptor is determined by conformational equilibrium in the transmembrane region. Nat. Commun. 3, 1045. (18) Nygaard, R., Zou, Y., Dror, R. O., Mildorf, T. J., Arlow, D. H., Manglik, A., Pan, A. C., Liu, C. W., Fung, J. J., Bokoch, M. P., Thian, F. S., Kobilka, T. S., Shaw, D. E., Mueller, L., Prosser, R. S., and Kobilka, B. K. (2013) The Dynamic Process of β2-Adrenergic Receptor Activation. Cell 152, 532−542. (19) Sounier, R., Mas, C., Steyaert, J., Laeremans, T., Manglik, A., Huang, W., Kobilka, B. K., Déméné, H., and Granier, S. (2015) Propagation of conformational changes during μ-opioid receptor activation. Nature 524, 375−378. (20) Kim, T. H., Chung, K. Y., Manglik, A., Hansen, A. L., Dror, R. O., Mildorf, T. J., Shaw, D. E., Kobilka, B. K., and Prosser, R. S. (2013) The Role of Ligands on the Equilibria Between Functional States of a G Protein-Coupled Receptor. J. Am. Chem. Soc. 135, 9465−9474. (21) Manglik, A., Kim, T. H., Masureel, M., Altenbach, C., Yang, Z., Hilger, D., Lerch, M. T., Kobilka, T. S., Thian, F. S., Hubbell, W. L., Prosser, R. S., and Kobilka, B. K. (2015) Structural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling. Cell 161, 1101−1111. (22) Ye, L., Van Eps, N., Zimmer, M., Ernst, O. P., and Prosser, R. S. (2016) Activation of the A2A adenosine G-protein-coupled receptor by conformational selection. Nature 533, 265−268. (23) Van Eps, N., Caro, L. N., Morizumi, T., Kusnetzow, A. K., Szczepek, M., Hofmann, K. P., Bayburt, T. H., Sligar, S. G., Ernst, O. P., and Hubbell, W. L. (2017) Conformational equilibria of light-
agonists with extremely slow unbinding kinetics, such as aripiprazole, buprenorphine, and xanomeline, could act by trapping one or more of the transition states. Second, if the rate of transition from receptor:GαGDPβγ to receptor:Gα0βγ was slow, this would provide a mechanism to select against activation of noncognate G proteins and help to explain why selectivity determinants have been hard to identify40 and also why there may be cooperativity for cognate G protein activation in the presence of noncognate G proteins.41 Third, a kinetic mechanism would provide a means to allow coupling to G proteins to precede coupling to β-arrestins; this could occur if the G protein coupling kinetics had fast association/ dissociation with slow association/dissociation for β-arrestin interactions. In this case, even with a similar affinity of the agonist bound receptor for G proteins and β-arrestins, initial receptor occupancy would be dominated by G proteins with these interactions being replaced by those with β-arrestins over time. One of the most important questions that will need to be addressed is whether conformational changes along a reaction coordinate pathway are coupled for receptor and G protein, as appears to be the case for all-trans-retinal and transducin release. If this is the case, there are significant implications for the role of agonists in funnelling coupled conformational changes along the reaction pathway.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Sebastian George Barton Furness: 0000-0001-8655-8221 Author Contributions #
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS S.G.B.F. is an ARC future fellow (FT180100543). REFERENCES
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DOI: 10.1021/acsptsci.9b00054 ACS Pharmacol. Transl. Sci. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsptsci.9b00054 ACS Pharmacol. Transl. Sci. XXXX, XXX, XXX−XXX
