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Conformational Transitions and the Activation of Heterotrimeric G Proteins by G Protein-Coupled Receptors
- Christopher Draper-Joyce*Christopher Draper-Joyce*E-mail: [email protected]Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, AustraliaMore by Christopher Draper-Joyce
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- Sebastian George Barton Furness*Sebastian George Barton Furness*E-mail: [email protected]Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, AustraliaMore by Sebastian George Barton Furness
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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.
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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 long-time-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 high-energy 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.
A Reaction Coordinate Pathway
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The role of a GPCR is to act as an agonist-dependent activator of heterotrimeric G proteins (henceforth G proteins) and non-G 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 10-fold 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 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 receptor-bound, 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”.
Figure 1
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).
Figure 2
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).
Figure 3
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).
Conformational Transitions at the Level of the Receptor
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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 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 19F (20−22) or nitroxide spin labeling (21,23) of cysteine residues and approaches using fluorescent modifications relying either on changes in intrinsic fluorescence (24) dependent on local environment or on Förster 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 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 nanobody (26) 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 Prosser’s laboratory (20) 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-G0 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.
Conformational Transitions of the G Protein
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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,and30)), 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 β-phosphate 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 (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 receptor (16) 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.
Figure 4
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)
Figure 5
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)
Implications of the Model
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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 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 identify (40) 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.
Author Information
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- Sebastian George Barton Furness, Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia,
http://orcid.org/0000-0001-8655-8221,
References
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- 7Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) The 2.0 A crystal structure of a heterotrimeric G protein. Nature 379, 311– 319, DOI: 10.1038/379311a0[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XotVOnug%253D%253D&md5=707c6411553692d34216bc1b6b50c911The 2.0 Å crystal structure of a heterotrimeric G proteinLambright, David G.; Sondek, John; Bohm, Andrew; Skiba, Nikolai P.; Hamm, Heidi E.; Sigler, Paul B.Nature (London) (1996), 379 (6563), 311-19CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The structure of a heterotrimeric G protein (transducin) reveals the mechanism of the nucleotide-dependent engagement of the α and βγ subunits that regulates their interaction with receptor and effector mols. The interaction involves 2 distinct interfaces and dramatically alters the conformation of the α but not of the βγ subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor.
- 8Noel, 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, DOI: 10.1038/366654a0[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXntFyktg%253D%253D&md5=29e5757bc8f6664bbd4839082b9d02d2The 2.2 Å crystal structure of transducin-α complexed with GTPγSNoel, Joseph P.; Hamm, Heidi E.; Sigler, Paul B.Nature (London, United Kingdom) (1993), 366 (6456), 654-63CODEN: NATUAS; ISSN:0028-0836.The 2.2 Å crystal structure of the activated α-subunit of rod transducin, Gtα·GTPγS, shows the bound GTPγS mol. deep in a cleft between a domain structurally homologous to small GTPases and a helical domain unique to heterotrimeric G proteins. The structure, when combined with biochem. and genetic studies, suggests: how an activated receptor might open this cleft to allow nucleotide exchange; a mechanism for GTP-induced changes in effector and receptor binding surfaces; and a mechanism for GTPase activity not evident from previous data.
- 9Coleman, 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, DOI: 10.1126/science.8073283[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXntFGhtA%253D%253D&md5=947d2c3671d0ae6764d451b08313a153Structures of active conformations of Giα1 and the mechanism of GTP hydrolysisColeman, David E.; Berghuis, Albert M.; Lee, Ethan; Linder, Maurine E.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, DC, United States) (1994), 265 (5177), 1405-12CODEN: SCIEAS; ISSN:0036-8075.Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein α subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resoln. x-ray structures of the GTPγS and GDP·AlF4- complexes formed by the G protein Giα1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the neg. charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the Gα family, this residue may account for the higher hydrolytic rate of Gα proteins relative to those of the p21ras family members. The fold of Giα1 differs from that of the homologous Gtα subunit in the conformation of a helix-loop sequence located in the α-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTPγS-Giα1, suggesting a mechanism that may promote release of the βγ subunit complex when the α subunit is activated by GTP.
- 10Sunahara, 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, DOI: 10.1126/science.278.5345.1943[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXotVClsbs%253D&md5=8d4552a0cb251ef0ad384be1376f27acCrystal structure of the adenylyl cyclase activator GsαSunahara, Roger K.; Tesmer, John J. G.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, D. C.) (1997), 278 (5345), 1943-1947CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The crystal structure of Gsα, the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was detd. at 2.5 Å in a complex with guanosine 5'-O-(3-thiotriphosphate)(GTPγS). Gsα is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsα·GTPγS with that of Giα·GTPγS suggested that their effector specificity was primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homol. of these elements. In contrast, sequence divergence explained the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsα. The βγ binding surface of Gsα was largely conserved in sequence and structure to that of Giα, whereas differences in the surface formed by the C-terminal helix and the α4-β6 loop may mediate receptor specificity.
- 11Rasmussen, 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, DOI: 10.1038/nature10361[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1equrrL&md5=d22a43dd677ac255d138b1aedff357d3Crystal structure of the β2 adrenergic receptor-Gs protein complexRasmussen, Soren G. F.; DeVree, Brian T.; Zou, Yao-Zhong; Kruse, Andrew C.; Chung, Ka-Young; Kobilka, Tong-Sun; Thian, Foon-Sun; Chae, Pil-Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M.; Shah, Syed T. A.; Lyons, Joseph A.; Caffrey, Martin; Gellman, Samuel H.; Steyaert, Jan; Skiniotis, Georgios; Weis, William I.; Sunahara, Roger K.; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 477 (7366), 549-555CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helixes of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resoln. view of transmembrane signalling by a GPCR.
- 12Liang, 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, DOI: 10.1038/nature22327[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslehs7w%253D&md5=cf34886db58191741437168ac3256c03Phase-plate cryo-EM structure of a class B GPCR-G-protein complexLiang, Yi-Lynn; Khoshouei, Maryam; Radjainia, Mazdak; Zhang, Yan; Glukhova, Alisa; Tarrasch, Jeffrey; Thal, David M.; Furness, Sebastian G. B.; Christopoulos, George; Coudrat, Thomas; Danev, Radostin; Baumeister, Wolfgang; Miller, Laurence J.; Christopoulos, Arthur; Kobilka, Brian K.; Wootten, Denise; Skiniotis, Georgios; Sexton, Patrick M.Nature (London, United Kingdom) (2017), 546 (7656), 118-123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Class B G-protein-coupled receptors (GPCRs) are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes, and obesity. Here, we report the structure of a full-length class B GPCR receptor, the calcitonin receptor, in complex with the peptide ligand, calcitonin, and a heterotrimeric Gαsβγ protein detd. by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist, calcitonin, engaged the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helixes 6 and 7. This conformation was accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also obsd. was an extended intracellular helix 8 that contributed to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. Thus, this structure provides a new framework for understanding GPCR receptor function.
- 13Draper-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, DOI: 10.1038/s41586-018-0236-6[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFOrsL3P&md5=f31a757364c3ff512bd5548b31e7f1bdStructure of the adenosine-bound human adenosine A1 receptor-Gi complexDraper-Joyce, Christopher J.; Khoshouei, Maryam; Thal, David M.; Liang, Yi-Lynn; Nguyen, Anh T. N.; Furness, Sebastian G. B.; Venugopal, Hariprasad; Baltos, Jo-Anne; Plitzko, Jurgen M.; Danev, Radostin; Baumeister, Wolfgang; May, Lauren T.; Wootten, Denise; Sexton, Patrick M.; Glukhova, Alisa; Christopoulos, ArthurNature (London, United Kingdom) (2018), 558 (7711), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Research)The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein detd. by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides mol. insights into receptor and G-protein selectivity.
- 14Mixon, 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, DOI: 10.1126/science.270.5238.954[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlOltbk%253D&md5=ebadfc21fc2b85d72b2ceccf2a150ff3Tertiary and quaternary structural changes in Giα1 induced by GTP hydrolysisMixon, Mark B.; Lee, Ethan; Coleman, David E.; Berghuis, Albert M.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, D. C.) (1995), 270 (5238), 954-60CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Crystallog. anal. of 2.2 angstrom resoln. shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein α subunit, Giα1. The switch II and switch III segments become disordered, and linker II connecting the Ras and α helical domains moves, thus altering the structures of potential effector and βγ binding regions. Contacts between the α-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and βγ binding determinants, are disordered in the complex with GTP, but are organized in to a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring α subunits in the lattice, suggesting that multimers of α subunits or heterotrimers may play a role in signal transduction.
- 15Roberts, 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, DOI: 10.1016/j.bcp.2004.05.044[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Kmt7w%253D&md5=4c6ebf4a4750a87f53aca787f5d1ff44G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?Roberts, David J.; Waelbroeck, MagaliBiochemical Pharmacology (2004), 68 (5), 799-806CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)A review. G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymic model of G protein activation and compares its predictions with the ternary complex model and with obsd. receptor phenomenon. This alternative model does not merely provide a new set of formulas but leads to a new philosophical outlook and more readily accommodates exptl. observations. The ternary complex model implies that, agonist-receptor-G-protein (HRG) being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).
- 16Furness, 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, DOI: 10.1016/j.cell.2016.09.021[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Clt7vI&md5=637ca081944a34b940df8b127a3dc669Ligand-Dependent Modulation of G Protein Conformation Alters Drug EfficacyFurness, Sebastian George Barton; Liang, Yi-Lynn; Nowell, Cameron James; Halls, Michelle Louise; Wookey, Peter John; Dal Maso, Emma; Inoue, Asuka; Christopoulos, Arthur; Wootten, Denise; Sexton, Patrick MichaelCell (Cambridge, MA, United States) (2016), 167 (3), 739-749.e11CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a mol. level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the mol. basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism.
- 17Kofuku, Y., Ueda, T., Okude, J., Shiraishi, Y., Kondo, K., Maeda, M., Tsujishita, H., and Shimada, I. (2012) Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region. Nat. Commun. 3, 1045, DOI: 10.1038/ncomms2046[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38bktFamtA%253D%253D&md5=ae8823d166374fe41851b214f8f461f3Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane regionKofuku Yutaka; Ueda Takumi; Okude Junya; Shiraishi Yutaro; Kondo Keita; Maeda Masahiro; Tsujishita Hideki; Shimada IchioNature communications (2012), 3 (), 1045 ISSN:.Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β(2)-adrenergic receptor (β(2)AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β(2)AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.
- 18Nygaard, 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, DOI: 10.1016/j.cell.2013.01.008[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFaiu7s%253D&md5=8f9aaa581027657b166b92ef617e950dThe Dynamic Process of β2-Adrenergic Receptor ActivationNygaard, Rie; Zou, Yaozhong; Dror, Ron O.; Mildorf, Thomas J.; Arlow, Daniel H.; Manglik, Aashish; Pan, Albert C.; Liu, Corey W.; Fung, Juan Jose; Bokoch, Michael P.; Thian, Foon Sun; Kobilka, Tong Sun; Shaw, David E.; Mueller, Luciano; Prosser, R. Scott; Kobilka, Brian K.Cell (Cambridge, MA, United States) (2013), 152 (3), 532-542CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, the authors use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β2-adrenergic receptor (β2AR), a prototypical GPCR. The authors labeled β2AR with 13CH3ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not obsd. in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound prepns. They also show that for β2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The obsd. heterogeneity may be important for β2AR's ability to engage multiple signaling and regulatory proteins.
- 19Sounier, 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, DOI: 10.1038/nature14680[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht12jtb%252FN&md5=0dd0dedcce583a52b4adc3084726a862Propagation of conformational changes during μ-opioid receptor activationSounier, Remy; Mas, Camille; Steyaert, Jan; Laeremans, Toon; Manglik, Aashish; Huang, Weijiao; Kobilka, Brian K.; Demene, Helene; Granier, SebastienNature (London, United Kingdom) (2015), 524 (7565), 375-378CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)μ-Opioid receptors (μORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (W. Huang et al., 2015) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use soln.-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) prepn. in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU 72 alone, or with BU 72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that obsd. for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.
- 20Kim, 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, DOI: 10.1021/ja404305k[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosFSqt74%253D&md5=29a622e3edde4e3cf9f29fe0fcb785b1The role of ligands on the equilibria between functional states of a G protein-coupled receptorKim, Tae Hun; Chung, Ka Young; Manglik, Aashish; Hansen, Alexandar L.; Dror, Ron O.; Mildorf, Thomas J.; Shaw, David E.; Kobilka, Brian K.; Prosser, R. ScottJournal of the American Chemical Society (2013), 135 (25), 9465-9474CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), 19F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed 2 distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Anal. of the spectra as a function of temp. revealed that for all ligands, the activation intermediate was entropically favored and enthalpically disfavored. The β2AR enthalpy changes toward activation were notably lower than those obsd. with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR were weaker. Pos. entropy changes toward activation likely reflected greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter anal. Ligands greatly influenced the overall changes in the enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.
- 21Manglik, 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, DOI: 10.1016/j.cell.2015.04.043[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosVaqsLw%253D&md5=aa8772ca46629b75b503d89f3ad0dd8dStructural Insights into the Dynamic Process of β2-Adrenergic Receptor SignalingManglik, Aashish; Kim, Tae Hun; Masureel, Matthieu; Altenbach, Christian; Yang, Zhongyu; Hilger, Daniel; Lerch, Michael T.; Kobilka, Tong Sun; Thian, Foon Sun; Hubbell, Wayne L.; Prosser, R. Scott; Kobilka, Brian K.Cell (Cambridge, MA, United States) (2015), 161 (5), 1101-1111CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equil. toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior obsd. for many GPCRs.
- 22Ye, 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, DOI: 10.1038/nature17668[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xnt1Ogsrc%253D&md5=d346565e2dcb9c96858554fb9f6d5085Activation of the A2A adenosine G-protein-coupled receptor by conformational selectionYe, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P.; Scott Prosser, R.Nature (London, United Kingdom) (2016), 533 (7602), 265-268CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Conformational selection and induced fit are two prevailing mechanisms to explain the mol. basis for ligand-based activation of receptors. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are important drug targets. A mol. understanding of their activation mechanism is crit. for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use 19F NMR to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G protein-coupled receptor. We find an ensemble of four states in equil.: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helixes 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addn. of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equil. of ligand-dependent G protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G protein-coupled receptor in health and disease.
- 23Van 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-activated rhodopsin in nanodiscs. Proc. Natl. Acad. Sci. U. S. A. 114, E3268– E3275, DOI: 10.1073/pnas.1620405114[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsV2nsLs%253D&md5=ad4a9c118a228c0a9f34402b62356dc4Conformational equilibria of light-activated rhodopsin in nanodiscsVan Eps, Ned; Caro, Lydia N.; Morizumi, Takefumi; Kusnetzow, Ana Karin; Szczepek, Michal; Hofmann, Klaus Peter; Bayburt, Timothy H.; Sligar, Stephen G.; Ernst, Oliver P.; Hubbell, Wayne L.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (16), E3268-E3275CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Conformational equil. of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here, conformational substates of the GPCR, rhodopsin, were investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helixes 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance (DEER) spectroscopy. The photoactivated receptor in DDM was dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is obsd., even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decayed to that characteristic of the inactive state with a lifetime of ∼16 min at 20°. Importantly, transducin binding to the activated receptor selected a subset of the ensemble in which multiple substates were apparently retained. The results indicated that in a native-like lipid environment rhodopsin activation was not analogous to a simple binary switch between 2 defined conformations, but the activated receptor was in equil. between multiple conformers that in principle could recognize different binding partners.
- 24Mary, S., Damian, M., Louet, M., Floquet, N., Fehrentz, J.-A., Marie, J., Martinez, J., and Baneres, J.-L. (2012) Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor. Proc. Natl. Acad. Sci. U. S. A. 109, 8304– 8309, DOI: 10.1073/pnas.1119881109[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XosFahsr4%253D&md5=4e8027be4f2606fa695d6bb7e30bc247Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptorMary, Sophie; Damian, Marjorie; Louet, Maxime; Floquet, Nicolas; Fehrentz, Jean-Alain; Marie, Jacky; Martinez, Jean; Baneres, Jean-LouisProceedings of the National Academy of Sciences of the United States of America (2012), 109 (21), 8304-8309, S8304/1-S8304/23CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid disks and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that obsd. in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacol. distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biol. responses. This is also likely to have major implications for the design of drugs activating specific GPCR-assocd. signaling pathways.
- 25Gregorio, G. G., Masureel, M., Hilger, D., Terry, D. S., Juette, M., Zhao, H., Zhou, Z., Perez-Aguilar, J. M., Hauge, M., Mathiasen, S., Javitch, J. A., Weinstein, H., Kobilka, B. K., and Blanchard, S. C. (2017) Single-molecule analysis of ligand efficacy in β2AR-G-protein activation. Nature 547, 68– 73, DOI: 10.1038/nature22354[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpt1Oqtb8%253D&md5=b7fc7f43e98bdf40611a7e7936b882afSingle-molecule analysis of ligand efficacy in β2AR-G-protein activationGregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel; Terry, Daniel S.; Juette, Manuel; Zhao, Hong; Zhou, Zhou; Perez-Aguilar, Jose Manuel; Hauge, Maria; Mathiasen, Signe; Javitch, Jonathan A.; Weinstein, Harel; Kobilka, Brian K.; Blanchard, Scott C.Nature (London, United Kingdom) (2017), 547 (7661), 68-73CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiol. and disease intervention, yet the mol. mechanisms responsible for ligand-dependent signaling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-mol. fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR-Gs species that are distinct from known structures, and provide single-mol. perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.
- 26Rasmussen, S., Choi, H., Fung, J., Pardon, E., Casarosa, P., Chae, P., DeVree, B., Rosenbaum, D., Thian, F., Kobilka, T. (2011) Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175– 180, DOI: 10.1038/nature09648[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkvFartA%253D%253D&md5=3a1d3bac6d92c9d54cf1ddd14d56ab8cStructure of a nanobody-stabilized active state of the β2 adrenoceptorRasmussen, Soren G. F.; Choi, Hee-Jung; Fung, Juan Jose; Pardon, Els; Casarosa, Paola; Chae, Pil Seok; DeVree, Brian T.; Rosenbaum, Daniel M.; Thian, Foon Sun; Kobilka, Tong Sun; Schnapp, Andreas; Konetzki, Ingo; Sunahara, Roger K.; Gellman, Samuel H.; Pautsch, Alexander; Steyaert, Jan; Weis, William I.; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 469 (7329), 175-180CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviors in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behavior, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are assocd. with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those obsd. in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.
- 27Schafer, C. T., Fay, J. F., Janz, J. M., and Farrens, D. L. (2016) Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state. Proc. Natl. Acad. Sci. U. S. A. 113, 11961– 11966, DOI: 10.1073/pnas.1606347113[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Srt7fI&md5=182075c0733d7899efe62eb9cac416a2Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor stateSchafer, Christopher T.; Fay, Jonathan F.; Janz, Jay M.; Farrens, David L.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (42), 11961-11966CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Here, the authors describe 2 insights into the role of receptor conformational dynamics during agonist release (all-trans retinal; ATR) from the visual G protein-coupled receptor (GPCR), rhodopsin. First, the authors show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, the authors obsd. that this equil. could be shifted by either promoting the active-like population or increasing the agonist concn. Second, the authors found that during decay of the signaling state an active-like, yet empty, receptor conformation could transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion was based on time-resolved, site-directed fluorescence labeling expts. that showed a small, but reproducible, lag between retinal leaving the protein and the return of transmembrane helix 6 (TM6) to the inactive conformation, as detd. from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissocn., either by addn. of hydroxylamine or by introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations showed that rhodopsin can bind its agonist in equil. like a traditional GPCR, provided evidence that an active GPCR conformation can persist even after agonist release, and raised the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.
- 28Kato, H. E., Zhang, Y., Hu, H., Suomivuori, C.-M., Kadji, F. M. N., Aoki, J., Krishna Kumar, K., Fonseca, R., Hilger, D., Huang, W., Latorraca, N. R., Inoue, A., Dror, R. O., Kobilka, B. K., and Skiniotis, G. (2019) Conformational transitions of a neurotensin receptor 1-Gi1 complex. Nature DOI: 10.1038/s41586-019-1337-6 . [Epub ahead of print].
- 29Heck, M. and Hofmann, K. P. (2001) Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism. J. Biol. Chem. 276, 10000– 10009, DOI: 10.1074/jbc.M009475200[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1OksLw%253D&md5=2987e5d6361cb577ab86e1969c595a5dMaximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation. Initial rate analysis based on a double displacement mechanismHeck, Martin; Hofmann, Klaus PeterJournal of Biological Chemistry (2001), 276 (13), 10000-10009CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, Gt, we have used kinetic light scattering, backed by direct biochem. assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the GtGDP substrate; (ii) to titrate GtGDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of Km = 2200 Gt/μm2 for GtGDP and Km = 230 μM for GTP; dissocn. consts. are Kd = 530 Gt/μm2 for R*-GtGDP dissocn. and Kd = 270 μM for GDP release from R*GtGDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 Gt/s at 22° and 1300 Gt/s at 34°. The anal. provides a tool to allocate and quantify better the effects of chem. or mutational protein modifications to individual steps in signal transduction.
- 30Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G., and Kobilka, B. K. (2019) Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes. Science 364, 552– 557, DOI: 10.1126/science.aaw5188[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXptFaks70%253D&md5=39d337fba0eff6fbb38bd8f2cbba3720Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexesMaeda, Shoji; Qu, Qianhui; Robertson, Michael J.; Skiniotis, Georgios; Kobilka, Brian K.Science (Washington, DC, United States) (2019), 364 (6440), 552-557CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/11 family, whereas M2R and M4R couple to the Gi/o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA. The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed anal. of these structures provides a framework for understanding the mol. determinants of G-protein coupling selectivity.
- 31Flock, T., Ravarani, C. N. J., Sun, D., Venkatakrishnan, A. J., Kayikci, M., Tate, C. G., Veprintsev, D. B., and Babu, M. M. (2015) Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173– 179, DOI: 10.1038/nature14663[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFyltLzF&md5=4eb6754be322f399caf2bbf831cec3ebUniversal allosteric mechanism for Gα activation by GPCRsFlock, Tilman; Ravarani, Charles N. J.; Sun, Dawei; Venkatakrishnan, A. J.; Kayikci, Melis; Tate, Christopher G.; Veprintsev, Dmitry B.; Babu, M. MadanNature (London, United Kingdom) (2015), 524 (7564), 173-179CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism.
- 32Posner, B. A., Mixon, M. B., Wall, M. A., Sprang, S. R., and Gilman, A. G. (1998) The A326S mutant of Gialpha1 as an approximation of the receptor bound state. J. Biol. Chem. 273, 21752– 21758, DOI: 10.1074/jbc.273.34.21752[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlsFOqs7c%253D&md5=58f0a3547839f3f02b7b6023bf63da1cThe A326S mutant of Giα1 as an approximation of the receptor-bound statePosner, Bruce A.; Mixon, Mark B.; Wall, Mark A.; Sprang, Stephen R.; Gilman, Alfred G.Journal of Biological Chemistry (1998), 273 (34), 21752-21758CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approxn. of the receptor-α subunit complex, we have considered the properties of A326S Giα1, a mutation discovered originally in Gsα (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissocn. of GDP from the αi1β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Giα1 for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissocn. from the heterotrimer (which is lost). For wild-type and mutant Giα1, βγ prevents irreversible inactivation of the α subunit at 30°. However, the mutation accelerates irreversible inactivation of α at 37° despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the no. of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those contg. A326S Giα1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers contg. A326S Giα1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissocn. of GDP can be obsd. in the absence of substantial structural alteration of a Gα subunit in the GDP-bound state.
- 33Brandt, D. R. and Ross, E. M. (1985) GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J. Biol. Chem. 260, 266– 272[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptF2ltw%253D%253D&md5=41ebfc79dbf08543f4f6e5fda12e4a4aGTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediatesBrandt, Douglas R.; Ross, Elliott M.Journal of Biological Chemistry (1985), 260 (1), 266-72CODEN: JBCHA3; ISSN:0021-9258.The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [α-32P]- or [γ-32P]GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTPγS) was used to quant. the total concn. of Gs. GTPase activity was a saturable function of the concn. of GTP, with a Km of 0.3 μM. MgCl2 monotonically increased the activity. The max. obsd. turnover no. was ∼ 1.5 min-1. During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t1/2 of ≤5 s at 30° and t1/2 ∼1 min at 0°. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concns. of either GTP or MgCl2 (>0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was const. at 0.3-0.4. The rate of dissocn. of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t1/2 of ∼ 1 min. Lubrol 12A9 potently inhibited the GTPase reaction and the dissocn. of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. The β and γ subunits of Gs markedly inhibited the dissocn. of GDP from Gs, in contrast to their ability to stimulate the dissocn. of GTPγS. GDP, GTPγS, and guanyl-5'-yl imidodiphosphate [Gpp(NH)p] competitively inhibited the accumulation of Gs-GDP. GTPγS and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and inorg. phosphate did not inhibit. Thus 2 specific mechanistic pathways for the overall GTPase reaction may coexist.
- 34Higashijima, T., Ferguson, K. M., Sternweis, P. C., Smigel, M. D., and Gilman, A. G. (1987) Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins. J. Biol. Chem. 262, 762– 766[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlartg%253D%253D&md5=c7868e70f8e76b0da863c19678340ff7Effects of magnesium and the βγ-subunit complex on the interactions of guanine nucleotides with G proteinsHigashijima, Tsutomu; Ferguson, Kenneth M.; Sternweis, Paul C.; Smigel, Murray D.; Gilman, Alfred G.Journal of Biological Chemistry (1987), 262 (2), 762-6CODEN: JBCHA3; ISSN:0021-9258.Mg2+ interacts with the α subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[γ-thio]triphosphate (GTPγS) to form a highly fluorescent complex from which nucleotide dissocs. very slowly. The apparent dissocn. const. for interaction of Gα·GTPγS with Mg2+ is ∼5 nM, similar to the Km for G protein GTPase activity. Gβγ increases the rate of dissocn. of GTPγS from Gα·GTPγS or Gα·GTPγS·Mg2+ at low Mg2+ concns. When the concn. of Mg2+ is >1 mM, Gβγ dissocs. from Gβγ·Gα·GTPγS·Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTPγS to Gα, the metal has relatively little effect on the binding of GDP. However, Bβγ increases the affinity of Gα for GDP by >100-fold. High concns. of Mg2+ promote the dissocn. of GDP from Gβγ·Gα·GDP, apparently without causing subunit dissocn. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissocn. of GDP from Gα under all conditions examd. Thus, there are ≥2 sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of Gβγ and GTPγS to Gα is neg. cooperative, whereas the binding interaction between Gβγ and GDP is strongly pos.
- 35Sun, X., Singh, S., Blumer, K., and Bowman, G. R. (2018) Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding. eLife 7, e38465, DOI: 10.7554/eLife.38465[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlyqsr7E&md5=9fb2812a94176f80c028c0b26751375cSimulation of spontaneous G protein activation reveals a new intermediate driving GDP unbindingSun, Xianqiang; Singh, Sukrit; Blumer, Kendall J.; Bowman, Gregory R.eLife (2018), 7 (), e38465/1-e38465/24CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ∼30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS anal. of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochem. data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. b- Strands 1 - 3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examg. unbinding processes with slow off-rates.
- 36Higashijima, T., Ferguson, K. M., Smigel, M. D., and Gilman, A. G. (1987) The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. J. Biol. Chem. 262, 757– 761[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlarsQ%253D%253D&md5=76ae6d8869065a77e884a6369f03e142The effect of GTP and magnesium on the GTPase activity and the fluorescent properties of GoHigashijima, Tsutomu; Ferguson, Kenneth M.; Smigel, Murray D.; Gilman, Alfred G.Journal of Biological Chemistry (1987), 262 (2), 757-61CODEN: JBCHA3; ISSN:0021-9258.GTP causes an increase in the fluorescence of Go, a G protein from bovine brain. When Mg2+ is also present, the increase in fluorescence is transient, and the rate of decline in the intensity of the fluorescence is the same as the rate of GTP hydrolysis by the protein. The steady-state rate of hydrolysis of GTP by Go (0.3-0.4/min) is slower than the catalytic rate of the protein (2/min), because the rate-limiting step in the reaction is the release of GDP.
- 37Du, Y., Duc, N. M., Rasmussen, S. G. F., Hilger, D., Kubiak, X., Wang, L., Bohon, J., Kim, H. R., Wegrecki, M., Asuru, A., Jeong, K. M., Lee, J., Chance, M. R., Lodowski, D. T., Kobilka, B. K., and Chung, K. Y. (2019) Assembly of a GPCR-G Protein Complex. Cell 177, 1232– 1242, DOI: 10.1016/j.cell.2019.04.022[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVOqtbk%253D&md5=7e1c310efcda7dcc2275955887273d09Assembly of a GPCR-G Protein ComplexDu, Yang; Duc, Nguyen Minh; Rasmussen, Soeren G. F.; Hilger, Daniel; Kubiak, Xavier; Wang, Liwen; Bohon, Jennifer; Kim, Hee Ryung; Wegrecki, Marcin; Asuru, Awuri; Jeong, Kyung Min; Lee, Jeongmi; Chance, Mark R.; Lodowski, David T.; Kobilka, Brian K.; Chung, Ka YoungCell (Cambridge, MA, United States) (2019), 177 (5), 1232-1242.e11CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallog. and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are obsd., there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is detd. by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes obsd. in crystal and cryo-EM structures.
- 38Roberts, D. J., Lin, H., and Strange, P. G. (2004) Mechanisms of agonist action at D2 dopamine receptors. Mol. Pharmacol. 66, 1573– 1579, DOI: 10.1124/mol.104.004077[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVOit7vJ&md5=6954bf6bdef1dc313e0d8b24464095eaMechanisms of agonist action at D2 dopamine receptorsRoberts, David J.; Lin, Hong; Strange, Philip G.Molecular Pharmacology (2004), 66 (6), 1573-1579CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)In this study, we investigated the biochem. mechanisms of agonist action at the G protein-coupled D2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding by full and partial agonists was detd. at different concns. of [35S]GTPγS (0.1 and 10 nM) and in the presence of different concns. of GDP. At both concns. of [35S]GTPγS, increasing GDP decreased the [35S]GTPγS binding obsd. with maximally stimulating concns. of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concns. Concn.-response expts. were performed for a range of agonists at the two [35S]GTPγS concns. and with different concns. of GDP. At 0.1 nM [35S]GTPγS, the potency of both full and partial agonists was dependent on the GDP concn. in the assays. At 10 nM [35S]GTPγS, the potency of full agonists exhibited a greater dependence on the GDP concn., whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [35S]GTPγS concn., the rate-detg. step in G protein activation is the binding of [35S]GTPγS to the G protein. At the higher [35S]GTPγS concn., for full agonists, [35S]GTPγS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-detg.
- 39Grant, B. J., McCammon, J. A., and Gorfe, A. A. (2010) Conformational selection in G-proteins: lessons from Ras and Rho. Biophys. J. 99, L87– 9, DOI: 10.1016/j.bpj.2010.10.020[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsV2js7fJ&md5=7fe80e3aab3fa9e57e7bb5e3c51860eaConformational selection in G-proteins: Lessons from Ras and RhoGrant, Barry J.; McCammon, J. Andrew; Gorfe, Alemayehu A.Biophysical Journal (2010), 99 (11), L87-L89CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The induced fit model has traditionally been invoked to describe the activating conformational change of monomeric G-proteins, such as Ras and Rho. With this scheme, the presence or absence of the γ-phosphate of GTP leads to an instantaneous switch in conformation. Here, the authors describe atomistic mol. simulations that demonstrate that both Ras and Rho superfamily members harbor an intrinsic susceptibility to sample multiple conformational states in the absence of nucleotide ligand. By comparing the distribution of conformers in the presence and absence of nucleotide, the authors show that conformational selection is the dominant mechanism by which Ras and Rho undergo nucleotide-dependent conformational changes. Furthermore, the pattern of correlated motions revealed by these simulations predicts a preserved allosteric coupling of the nucleotide-binding site with the membrane interacting C-terminus in both Rho and Ras.
- 40Flock, T., Hauser, A. S., Lund, N., Gloriam, D. E., Balaji, S., and Babu, M. M. (2017) Selectivity determinants of GPCR-G-protein binding. Nature 545, 317– 322, DOI: 10.1038/nature22070[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsVeis74%253D&md5=947db386e0e0f74b09465a99d880c518Selectivity determinants of GPCR-G-protein bindingFlock, Tilman; Hauser, Alexander S.; Lund, Nadia; Gloriam, David E.; Balaji, Santhanam; Babu, M. MadanNature (London, United Kingdom) (2017), 545 (7654), 317-322CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is crit. to trigger the appropriate physiol. response. However, the determinants of selective binding have remained elusive. Here, we reveal the existence of a selectivity barcode (i.e., patterns of amino acids) on each of the 16 human G proteins that is recognized by distinct regions on the ∼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 nonidentical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-detg. residues. These findings lay the foundation for understanding the mol. basis of coupling selectivity within individual receptors and G proteins.
- 41Gupte, T. M., Malik, R. U., Sommese, R. F., Ritt, M., and Sivaramakrishnan, S. (2017) Priming GPCR signaling through the synergistic effect of two G proteins. Proc. Natl. Acad. Sci. U. S. A. 114, 3756– 3761, DOI: 10.1073/pnas.1617232114[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXks1Smtrk%253D&md5=d3d4c469231ebfef8e908e28199c0b8dPriming GPCR signaling through the synergistic effect of two G proteinsGupte, Tejas M.; Malik, Rabia U.; Sommese, Ruth F.; Ritt, Michael; Sivaramakrishnan, SivarajProceedings of the National Academy of Sciences of the United States of America (2017), 114 (14), 3756-3761CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimol. event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming.". Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asym. GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.
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Figure 1
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).
Figure 2
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).
Figure 3
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).
Figure 4
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)
Figure 5
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)
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- 3Agarwal, P. K., Doucet, N., Chennubhotla, C., Ramanathan, A., and Narayanan, C. (2016) Conformational Sub-states and Populations in Enzyme Catalysis. Methods Enzymol. 578, 273– 297, DOI: 10.1016/bs.mie.2016.05.023[Crossref], [PubMed], [CAS], Google Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC2s3otFSgtg%253D%253D&md5=76ae795549bf89c09585819609db6f2fConformational Sub-states and Populations in Enzyme CatalysisAgarwal P K; Doucet N; Narayanan C; Chennubhotla C; Ramanathan AMethods in enzymology (2016), 578 (), 273-97 ISSN:.Enzyme function involves substrate and cofactor binding, precise positioning of reactants in the active site, chemical turnover, and release of products. In addition to formation of crucial structural interactions between enzyme and substrate(s), coordinated motions within the enzyme-substrate complex allow reaction to proceed at a much faster rate, compared to the reaction in solution and in the absence of enzyme. An increasing number of enzyme systems show the presence of conserved protein motions that are important for function. A wide variety of motions are naturally sampled (over femtosecond to millisecond time-scales) as the enzyme complex moves along the energetic landscape, driven by temperature and dynamical events from the surrounding environment. Areas of low energy along the landscape form conformational sub-states, which show higher conformational populations than surrounding areas. A small number of these protein conformational sub-states contain functionally important structural and dynamical features, which assist the enzyme mechanism along the catalytic cycle. Identification and characterization of these higher-energy (also called excited) sub-states and the associated populations are challenging, as these sub-states are very short-lived and therefore rarely populated. Specialized techniques based on computer simulations, theoretical modeling, and nuclear magnetic resonance have been developed for quantitative characterization of these sub-states and populations. This chapter discusses these techniques and provides examples of their applications to enzyme systems.
- 4De Lean, A., Stadel, J. M., and Lefkowitz, R. J. (1980) A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupledbeta-adrenergic receptor. J. Biol. Chem. 255 (15), 7108– 17[Crossref], [PubMed], [CAS], Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL3cXlt1KjsL4%253D&md5=8e88d602b3617ffe1c80cffef3f299c1A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled β-adrenergic receptorDe Lean, Andre; Stadel, Jeffrey M.; Lefkowitz, Robert J.Journal of Biological Chemistry (1980), 255 (15), 7108-17CODEN: JBCHA3; ISSN:0021-9258.The unique properties of agonist binding to the frog erythrocyte β-adrenergic receptor include the existence of 2 affinity forms of the receptor. The proportion and relative affinity of these 2 states of the receptor for ligands varies with the intrinsic activity of the agonist and the presence of guanine nucleotides. The simplest model for hormone-receptor interactions which can explain and reproduce the exptl. data involves the interaction of the receptor R with an addnl. membrane component X, leading to the agonist-promoted formation of a high affinity ternary complex HRX. Computer modeling of agonist binding data with a ternary complex model indicates that the model can fit the data with high accuracy under conditions where the ligand used is either a full or a partial agonist and where the system is altered by the addn. of guanine nucleotide or after treatment with group-specific reagents, e.g., p-hydroxymercuribenzoate. The parameter ests. obtained indicated that the intrinsic activity of the agonist is correlated with the affinity const. L of the component X for the binary complex HR. The major effect of adding guanine nucleotides is to destabilize the ternary complex HRX from which both the hormone H and the component X can dissoc. The modulatory role of nucleotides on the affinity of agonists for the receptor is consistent with the assumption that the component X is the guanine nucleotide binding site. The ternary complex model was also applied successfully to the turkey erythrocyte receptor system. The model provides a general scheme for the activation by agonists of adenylate cyclase [9012-42-4]-coupled receptor systems and also of other systems where the effector might be different.
- 5Glukhova, A., Draper-Joyce, C., Sunahara, R. K., Christopoulos, A., Wootten, D., and Sexton, P. M. (2018) Rules of engagement: GPCRs and G proteins. ACS Pharmacology & Translational Science 1, 73– 83, DOI: 10.1021/acsptsci.8b00026[ACS Full Text], [CAS], Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhs1Klt7vF&md5=016702bca92d44aeeff39162b94d6d38Rules of Engagement: GPCRs and G ProteinsGlukhova, Alisa; Draper-Joyce, Christopher J.; Sunahara, Roger K.; Christopoulos, Arthur; Wootten, Denise; Sexton, Patrick M.ACS Pharmacology & Translational Science (2018), 1 (2), 73-83CODEN: APTSFN; ISSN:2575-9108. (American Chemical Society)A review. G protein-coupled receptors (GPCRs) are a key drug target class. They account for over one third of current pharmaceuticals, and both drugs that inhibit and promote receptor function are important therapeutically; in some cases, the same GPCR can be targeted with agonists and inhibitors, depending upon disease context. There have been major breakthroughs in understanding GPCR structure and drug binding through advances in x-ray crystallog., and membrane protein stabilization. Nonetheless, these structures have predominately been of inactive receptors bound to inhibitors. Efforts to capture structures of fully active GPCRs, in particular those in complex with the canonical, physiol. transducer G protein, have been limited via this approach. Very recently, advances in cryo-electron microscopy have provided access to agonist:GPCR:G protein complex structures. These promise to revolutionize our understanding of GPCR:G protein engagement and provide insight into mechanisms of efficacy, coupling selectivity and how these might be controlled by biased agonists. Here we review what we have currently learned from the new GPCR:Gs and GPCR:Gi/o complex structures.
- 6Wall, M. A., Coleman, D. E., Lee, E., Iñiguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) The structure of the G protein heterotrimerGi alpha 1 beta 1 gamma 2. Cell 83, 1047– 1058, DOI: 10.1016/0092-8674(95)90220-1[Crossref], [PubMed], [CAS], Google Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXhtVSnu7fK&md5=205599b12da8a67d97681e2b66f33ecdThe structure of the G protein heterotrimer Giα1β1γ2Wall, Mark A.; Coleman, David E.; Lee, Ethan; Iniguez-Lluhi, Jorge A.; Posner, Bruce A.; Gilman, Alfred G.; Sprang, Stephen R.Cell (Cambridge, Massachusetts) (1995), 83 (6), 1047-58CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The crystallog. structure of the G protein heterotrimer Giα1(GDP)β1γ2 (at 2.3 Å) reveals two nonoverlapping regions of contact between α and β, an extended interface between β and nearly all of γ, and limited interaction of α with γ. The major α/β interface covers switch II of α, and GTP-induced rearrangement of switch II causes subunit dissocn. during signaling. Alterations in GDP binding in the heterotrimer (compared with α-GDP) explain stabilization of the inactive conformation of α by βγ. Repeated WD motifs in β form a circularized sevenfold β propeller. The conserved cores of these motifs are a scaffold for display of their more variable linkers on the exterior face of each propeller blade.
- 7Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) The 2.0 A crystal structure of a heterotrimeric G protein. Nature 379, 311– 319, DOI: 10.1038/379311a0[Crossref], [PubMed], [CAS], Google Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK28XotVOnug%253D%253D&md5=707c6411553692d34216bc1b6b50c911The 2.0 Å crystal structure of a heterotrimeric G proteinLambright, David G.; Sondek, John; Bohm, Andrew; Skiba, Nikolai P.; Hamm, Heidi E.; Sigler, Paul B.Nature (London) (1996), 379 (6563), 311-19CODEN: NATUAS; ISSN:0028-0836. (Macmillan Magazines)The structure of a heterotrimeric G protein (transducin) reveals the mechanism of the nucleotide-dependent engagement of the α and βγ subunits that regulates their interaction with receptor and effector mols. The interaction involves 2 distinct interfaces and dramatically alters the conformation of the α but not of the βγ subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor.
- 8Noel, 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, DOI: 10.1038/366654a0[Crossref], [PubMed], [CAS], Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2cXntFyktg%253D%253D&md5=29e5757bc8f6664bbd4839082b9d02d2The 2.2 Å crystal structure of transducin-α complexed with GTPγSNoel, Joseph P.; Hamm, Heidi E.; Sigler, Paul B.Nature (London, United Kingdom) (1993), 366 (6456), 654-63CODEN: NATUAS; ISSN:0028-0836.The 2.2 Å crystal structure of the activated α-subunit of rod transducin, Gtα·GTPγS, shows the bound GTPγS mol. deep in a cleft between a domain structurally homologous to small GTPases and a helical domain unique to heterotrimeric G proteins. The structure, when combined with biochem. and genetic studies, suggests: how an activated receptor might open this cleft to allow nucleotide exchange; a mechanism for GTP-induced changes in effector and receptor binding surfaces; and a mechanism for GTPase activity not evident from previous data.
- 9Coleman, 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, DOI: 10.1126/science.8073283[Crossref], [PubMed], [CAS], Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXntFGhtA%253D%253D&md5=947d2c3671d0ae6764d451b08313a153Structures of active conformations of Giα1 and the mechanism of GTP hydrolysisColeman, David E.; Berghuis, Albert M.; Lee, Ethan; Linder, Maurine E.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, DC, United States) (1994), 265 (5177), 1405-12CODEN: SCIEAS; ISSN:0036-8075.Mechanisms of guanosine triphosphate (GTP) hydrolysis by members of the G protein α subunit-p21ras superfamily of guanosine triphosphatases have been studied extensively but have not been well understood. High-resoln. x-ray structures of the GTPγS and GDP·AlF4- complexes formed by the G protein Giα1 demonstrate specific roles in transition-state stabilization for two highly conserved residues. Glutamine204 (Gln61 in p21ras) stabilizes and orients the hydrolytic water in the trigonal-bipyramidal transition state. Arginine 178 stabilizes the neg. charge at the equatorial oxygen atoms of the pentacoordinate phosphate intermediate. Conserved only in the Gα family, this residue may account for the higher hydrolytic rate of Gα proteins relative to those of the p21ras family members. The fold of Giα1 differs from that of the homologous Gtα subunit in the conformation of a helix-loop sequence located in the α-helical domain that is characteristic of these proteins; this site may participate in effector binding. The amino-terminal 33 residues are disordered in GTPγS-Giα1, suggesting a mechanism that may promote release of the βγ subunit complex when the α subunit is activated by GTP.
- 10Sunahara, 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, DOI: 10.1126/science.278.5345.1943[Crossref], [PubMed], [CAS], Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2sXotVClsbs%253D&md5=8d4552a0cb251ef0ad384be1376f27acCrystal structure of the adenylyl cyclase activator GsαSunahara, Roger K.; Tesmer, John J. G.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, D. C.) (1997), 278 (5345), 1943-1947CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)The crystal structure of Gsα, the heterotrimeric G protein α subunit that stimulates adenylyl cyclase, was detd. at 2.5 Å in a complex with guanosine 5'-O-(3-thiotriphosphate)(GTPγS). Gsα is the prototypic member of a family of GTP-binding proteins that regulate the activities of effectors in a hormone-dependent manner. Comparison of the structure of Gsα·GTPγS with that of Giα·GTPγS suggested that their effector specificity was primarily dictated by the shape of the binding surface formed by the switch II helix and the α3-β5 loop, despite the high sequence homol. of these elements. In contrast, sequence divergence explained the inability of regulators of G protein signaling to stimulate the GTPase activity of Gsα. The βγ binding surface of Gsα was largely conserved in sequence and structure to that of Giα, whereas differences in the surface formed by the C-terminal helix and the α4-β6 loop may mediate receptor specificity.
- 11Rasmussen, 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, DOI: 10.1038/nature10361[Crossref], [PubMed], [CAS], Google Scholar11https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXht1equrrL&md5=d22a43dd677ac255d138b1aedff357d3Crystal structure of the β2 adrenergic receptor-Gs protein complexRasmussen, Soren G. F.; DeVree, Brian T.; Zou, Yao-Zhong; Kruse, Andrew C.; Chung, Ka-Young; Kobilka, Tong-Sun; Thian, Foon-Sun; Chae, Pil-Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M.; Shah, Syed T. A.; Lyons, Joseph A.; Caffrey, Martin; Gellman, Samuel H.; Steyaert, Jan; Skiniotis, Georgios; Weis, William I.; Sunahara, Roger K.; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 477 (7366), 549-555CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor (β2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helixes of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14 Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resoln. view of transmembrane signalling by a GPCR.
- 12Liang, 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, DOI: 10.1038/nature22327[Crossref], [PubMed], [CAS], Google Scholar12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXmslehs7w%253D&md5=cf34886db58191741437168ac3256c03Phase-plate cryo-EM structure of a class B GPCR-G-protein complexLiang, Yi-Lynn; Khoshouei, Maryam; Radjainia, Mazdak; Zhang, Yan; Glukhova, Alisa; Tarrasch, Jeffrey; Thal, David M.; Furness, Sebastian G. B.; Christopoulos, George; Coudrat, Thomas; Danev, Radostin; Baumeister, Wolfgang; Miller, Laurence J.; Christopoulos, Arthur; Kobilka, Brian K.; Wootten, Denise; Skiniotis, Georgios; Sexton, Patrick M.Nature (London, United Kingdom) (2017), 546 (7656), 118-123CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Class B G-protein-coupled receptors (GPCRs) are major targets for the treatment of chronic diseases, such as osteoporosis, diabetes, and obesity. Here, we report the structure of a full-length class B GPCR receptor, the calcitonin receptor, in complex with the peptide ligand, calcitonin, and a heterotrimeric Gαsβγ protein detd. by Volta phase-plate single-particle cryo-electron microscopy. The peptide agonist, calcitonin, engaged the receptor by binding to an extended hydrophobic pocket facilitated by the large outward movement of the extracellular ends of transmembrane helixes 6 and 7. This conformation was accompanied by a 60° kink in helix 6 and a large outward movement of the intracellular end of this helix, opening the bundle to accommodate interactions with the α5-helix of Gαs. Also obsd. was an extended intracellular helix 8 that contributed to both receptor stability and functional G-protein coupling via an interaction with the Gβ subunit. Thus, this structure provides a new framework for understanding GPCR receptor function.
- 13Draper-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, DOI: 10.1038/s41586-018-0236-6[Crossref], [PubMed], [CAS], Google Scholar13https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXhtFOrsL3P&md5=f31a757364c3ff512bd5548b31e7f1bdStructure of the adenosine-bound human adenosine A1 receptor-Gi complexDraper-Joyce, Christopher J.; Khoshouei, Maryam; Thal, David M.; Liang, Yi-Lynn; Nguyen, Anh T. N.; Furness, Sebastian G. B.; Venugopal, Hariprasad; Baltos, Jo-Anne; Plitzko, Jurgen M.; Danev, Radostin; Baumeister, Wolfgang; May, Lauren T.; Wootten, Denise; Sexton, Patrick M.; Glukhova, Alisa; Christopoulos, ArthurNature (London, United Kingdom) (2018), 558 (7711), 559-563CODEN: NATUAS; ISSN:0028-0836. (Nature Research)The class A adenosine A1 receptor (A1R) is a G-protein-coupled receptor that preferentially couples to inhibitory Gi/o heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human A1R in complex with adenosine and heterotrimeric Gi2 protein detd. by Volta phase plate cryo-electron microscopy. Compared to inactive A1R, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the A1R primarily via amino acids in the C terminus of the Gαi α5-helix, concomitant with a 10.5 Å outward movement of the A1R transmembrane domain 6. Comparison with the agonist-bound β2 adrenergic receptor-Gs-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active A1R structure provides mol. insights into receptor and G-protein selectivity.
- 14Mixon, 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, DOI: 10.1126/science.270.5238.954[Crossref], [PubMed], [CAS], Google Scholar14https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK2MXptlOltbk%253D&md5=ebadfc21fc2b85d72b2ceccf2a150ff3Tertiary and quaternary structural changes in Giα1 induced by GTP hydrolysisMixon, Mark B.; Lee, Ethan; Coleman, David E.; Berghuis, Albert M.; Gilman, Alfred G.; Sprang, Stephen R.Science (Washington, D. C.) (1995), 270 (5238), 954-60CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Crystallog. anal. of 2.2 angstrom resoln. shows that guanosine triphosphate (GTP) hydrolysis triggers conformational changes in the heterotrimeric G-protein α subunit, Giα1. The switch II and switch III segments become disordered, and linker II connecting the Ras and α helical domains moves, thus altering the structures of potential effector and βγ binding regions. Contacts between the α-helical and Ras domains are weakened, possibly facilitating the release of guanosine diphosphate (GDP). The amino and carboxyl termini, which contain receptor and βγ binding determinants, are disordered in the complex with GTP, but are organized in to a compact microdomain on GDP hydrolysis. The amino terminus also forms extensive quaternary contacts with neighboring α subunits in the lattice, suggesting that multimers of α subunits or heterotrimers may play a role in signal transduction.
- 15Roberts, 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, DOI: 10.1016/j.bcp.2004.05.044[Crossref], [PubMed], [CAS], Google Scholar15https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXmt1Kmt7w%253D&md5=4c6ebf4a4750a87f53aca787f5d1ff44G protein activation by G protein coupled receptors: ternary complex formation or catalyzed reaction?Roberts, David J.; Waelbroeck, MagaliBiochemical Pharmacology (2004), 68 (5), 799-806CODEN: BCPCA6; ISSN:0006-2952. (Elsevier B.V.)A review. G protein coupled receptors catalyze the GDP/GTP exchange on G proteins, thereby activating them. The ternary complex model, designed to describe agonist binding in the absence of GTP, is often extended to G protein activation. This is logically unsatisfactory as the ternary complex does not accumulate when G proteins are activated by GTP. Extended models taking into account nucleotide binding exist, but fail to explain catalytic G protein activation. This review puts forward an enzymic model of G protein activation and compares its predictions with the ternary complex model and with obsd. receptor phenomenon. This alternative model does not merely provide a new set of formulas but leads to a new philosophical outlook and more readily accommodates exptl. observations. The ternary complex model implies that, agonist-receptor-G-protein (HRG) being responsible for efficient G protein activation, it should be as stable as possible. In contrast, the enzyme model suggests that although a limited stabilization of HRG facilitates GDP release, HRG should not be "too stable" as this might trap the G protein in an inactive state and actually hinder G protein activation. The two models also differ completely in the definition of the receptor "active state": the ternary complex model implies that the active state corresponds to a single active receptor conformation (HRG); in contrast, the catalytic model predicts that the active receptor state is mobile, switching smoothly through various conformations with high and low affinities for agonists (HR, HRG, HRGGDP, HRGGTP, etc.).
- 16Furness, 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, DOI: 10.1016/j.cell.2016.09.021[Crossref], [PubMed], [CAS], Google Scholar16https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Clt7vI&md5=637ca081944a34b940df8b127a3dc669Ligand-Dependent Modulation of G Protein Conformation Alters Drug EfficacyFurness, Sebastian George Barton; Liang, Yi-Lynn; Nowell, Cameron James; Halls, Michelle Louise; Wookey, Peter John; Dal Maso, Emma; Inoue, Asuka; Christopoulos, Arthur; Wootten, Denise; Sexton, Patrick MichaelCell (Cambridge, MA, United States) (2016), 167 (3), 739-749.e11CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G protein-coupled receptor (GPCR) signaling, mediated by hetero-trimeric G proteins, can be differentially controlled by agonists. At a mol. level, this is thought to occur principally via stabilization of distinct receptor conformations by individual ligands. These distinct conformations control subsequent recruitment of transducer and effector proteins. Here, we report that ligand efficacy at the calcitonin GPCR (CTR) is also correlated with ligand-dependent alterations to G protein conformation. We observe ligand-dependent differences in the sensitivity of the G protein ternary complex to disruption by GTP, due to conformational differences in the receptor-bound G protein hetero-trimer. This results in divergent agonist-dependent receptor-residency times for the hetero-trimeric G protein and different accumulation rates for downstream second messengers. This study demonstrates that factors influencing efficacy extend beyond receptor conformation(s) and expands understanding of the mol. basis for how G proteins control/influence efficacy. This has important implications for the mechanisms that underlie ligand-mediated biased agonism.
- 17Kofuku, Y., Ueda, T., Okude, J., Shiraishi, Y., Kondo, K., Maeda, M., Tsujishita, H., and Shimada, I. (2012) Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane region. Nat. Commun. 3, 1045, DOI: 10.1038/ncomms2046[Crossref], [PubMed], [CAS], Google Scholar17https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A280%3ADC%252BC38bktFamtA%253D%253D&md5=ae8823d166374fe41851b214f8f461f3Efficacy of the β2-adrenergic receptor is determined by conformational equilibrium in the transmembrane regionKofuku Yutaka; Ueda Takumi; Okude Junya; Shiraishi Yutaro; Kondo Keita; Maeda Masahiro; Tsujishita Hideki; Shimada IchioNature communications (2012), 3 (), 1045 ISSN:.Many drugs that target G-protein-coupled receptors (GPCRs) induce or inhibit their signal transduction with different strengths, which affect their therapeutic properties. However, the mechanism underlying the differences in the signalling levels is still not clear, although several structures of GPCRs complexed with ligands determined by X-ray crystallography are available. Here we utilized NMR to monitor the signals from the methionine residue at position 82 in neutral antagonist- and partial agonist-bound states of β(2)-adrenergic receptor (β(2)AR), which are correlated with the conformational changes of the transmembrane regions upon activation. We show that this residue exists in a conformational equilibrium between the inverse agonist-bound states and the full agonist-bound state, and the population of the latter reflects the signal transduction level in each ligand-bound state. These findings provide insights into the multi-level signalling of β(2)AR and other GPCRs, including the basal activity, and the mechanism of signal transduction mediated by GPCRs.
- 18Nygaard, 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, DOI: 10.1016/j.cell.2013.01.008[Crossref], [PubMed], [CAS], Google Scholar18https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXhvFaiu7s%253D&md5=8f9aaa581027657b166b92ef617e950dThe Dynamic Process of β2-Adrenergic Receptor ActivationNygaard, Rie; Zou, Yaozhong; Dror, Ron O.; Mildorf, Thomas J.; Arlow, Daniel H.; Manglik, Aashish; Pan, Albert C.; Liu, Corey W.; Fung, Juan Jose; Bokoch, Michael P.; Thian, Foon Sun; Kobilka, Tong Sun; Shaw, David E.; Mueller, Luciano; Prosser, R. Scott; Kobilka, Brian K.Cell (Cambridge, MA, United States) (2013), 152 (3), 532-542CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, the authors use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of the β2-adrenergic receptor (β2AR), a prototypical GPCR. The authors labeled β2AR with 13CH3ε-methionine and obtained HSQC spectra of unliganded receptor as well as receptor bound to an inverse agonist, an agonist, and a G-protein-mimetic nanobody. These studies provide evidence for conformational states not obsd. in crystal structures, as well as substantial conformational heterogeneity in agonist- and inverse-agonist-bound prepns. They also show that for β2AR, unlike rhodopsin, an agonist alone does not stabilize a fully active conformation, suggesting that the conformational link between the agonist-binding pocket and the G-protein-coupling surface is not rigid. The obsd. heterogeneity may be important for β2AR's ability to engage multiple signaling and regulatory proteins.
- 19Sounier, 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, DOI: 10.1038/nature14680[Crossref], [PubMed], [CAS], Google Scholar19https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXht12jtb%252FN&md5=0dd0dedcce583a52b4adc3084726a862Propagation of conformational changes during μ-opioid receptor activationSounier, Remy; Mas, Camille; Steyaert, Jan; Laeremans, Toon; Manglik, Aashish; Huang, Weijiao; Kobilka, Brian K.; Demene, Helene; Granier, SebastienNature (London, United Kingdom) (2015), 524 (7565), 375-378CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)μ-Opioid receptors (μORs) are G-protein-coupled receptors that are activated by a structurally diverse spectrum of natural and synthetic agonists including endogenous endorphin peptides, morphine and methadone. The recent structures of the μOR in inactive and agonist-induced active states (W. Huang et al., 2015) provide snapshots of the receptor at the beginning and end of a signalling event, but little is known about the dynamic sequence of events that span these two states. Here we use soln.-state NMR to examine the process of μOR activation using a purified receptor (mouse sequence) prepn. in an amphiphile membrane-like environment. We obtain spectra of the μOR in the absence of ligand, and in the presence of the high-affinity agonist BU 72 alone, or with BU 72 and a G protein mimetic nanobody. Our results show that conformational changes in transmembrane segments 5 and 6 (TM5 and TM6), which are required for the full engagement of a G protein, are almost completely dependent on the presence of both the agonist and the G protein mimetic nanobody, revealing a weak allosteric coupling between the agonist-binding pocket and the G-protein-coupling interface (TM5 and TM6), similar to that obsd. for the β2-adrenergic receptor. Unexpectedly, in the presence of agonist alone, we find larger spectral changes involving intracellular loop 1 and helix 8 compared to changes in TM5 and TM6. These results suggest that one or both of these domains may play a role in the initial interaction with the G protein, and that TM5 and TM6 are only engaged later in the process of complex formation. The initial interactions between the G protein and intracellular loop 1 and/or helix 8 may be involved in G-protein coupling specificity, as has been suggested for other family A G-protein-coupled receptors.
- 20Kim, 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, DOI: 10.1021/ja404305k[ACS Full Text], [CAS], Google Scholar20https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3sXosFSqt74%253D&md5=29a622e3edde4e3cf9f29fe0fcb785b1The role of ligands on the equilibria between functional states of a G protein-coupled receptorKim, Tae Hun; Chung, Ka Young; Manglik, Aashish; Hansen, Alexandar L.; Dror, Ron O.; Mildorf, Thomas J.; Shaw, David E.; Kobilka, Brian K.; Prosser, R. ScottJournal of the American Chemical Society (2013), 135 (25), 9465-9474CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), 19F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl glycol detergent micelles revealed 2 distinct inactive states, an activation intermediate state en route to activation, and, in the presence of a G protein mimic, a predominant active state. Anal. of the spectra as a function of temp. revealed that for all ligands, the activation intermediate was entropically favored and enthalpically disfavored. The β2AR enthalpy changes toward activation were notably lower than those obsd. with rhodopsin, a likely consequence of basal activity and the fact that the ionic lock and other interactions stabilizing the inactive state of β2AR were weaker. Pos. entropy changes toward activation likely reflected greater mobility (configurational entropy) in the cytoplasmic domain, as confirmed through an order parameter anal. Ligands greatly influenced the overall changes in the enthalpy and entropy of the system and the corresponding changes in population and amplitude of motion of given states, suggesting a complex landscape of states and substates.
- 21Manglik, 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, DOI: 10.1016/j.cell.2015.04.043[Crossref], [PubMed], [CAS], Google Scholar21https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXosVaqsLw%253D&md5=aa8772ca46629b75b503d89f3ad0dd8dStructural Insights into the Dynamic Process of β2-Adrenergic Receptor SignalingManglik, Aashish; Kim, Tae Hun; Masureel, Matthieu; Altenbach, Christian; Yang, Zhongyu; Hilger, Daniel; Lerch, Michael T.; Kobilka, Tong Sun; Thian, Foon Sun; Hubbell, Wayne L.; Prosser, R. Scott; Kobilka, Brian K.Cell (Cambridge, MA, United States) (2015), 161 (5), 1101-1111CODEN: CELLB5; ISSN:0092-8674. (Cell Press)G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the β2-adrenergic receptor (β2AR) using 19F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound β2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equil. toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior obsd. for many GPCRs.
- 22Ye, 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, DOI: 10.1038/nature17668[Crossref], [PubMed], [CAS], Google Scholar22https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xnt1Ogsrc%253D&md5=d346565e2dcb9c96858554fb9f6d5085Activation of the A2A adenosine G-protein-coupled receptor by conformational selectionYe, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P.; Scott Prosser, R.Nature (London, United Kingdom) (2016), 533 (7602), 265-268CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)Conformational selection and induced fit are two prevailing mechanisms to explain the mol. basis for ligand-based activation of receptors. G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors and are important drug targets. A mol. understanding of their activation mechanism is crit. for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use 19F NMR to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G protein-coupled receptor. We find an ensemble of four states in equil.: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helixes 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addn. of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equil. of ligand-dependent G protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G protein-coupled receptor in health and disease.
- 23Van 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-activated rhodopsin in nanodiscs. Proc. Natl. Acad. Sci. U. S. A. 114, E3268– E3275, DOI: 10.1073/pnas.1620405114[Crossref], [PubMed], [CAS], Google Scholar23https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXlsV2nsLs%253D&md5=ad4a9c118a228c0a9f34402b62356dc4Conformational equilibria of light-activated rhodopsin in nanodiscsVan Eps, Ned; Caro, Lydia N.; Morizumi, Takefumi; Kusnetzow, Ana Karin; Szczepek, Michal; Hofmann, Klaus Peter; Bayburt, Timothy H.; Sligar, Stephen G.; Ernst, Oliver P.; Hubbell, Wayne L.Proceedings of the National Academy of Sciences of the United States of America (2017), 114 (16), E3268-E3275CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Conformational equil. of G-protein-coupled receptors (GPCRs) are intimately involved in intracellular signaling. Here, conformational substates of the GPCR, rhodopsin, were investigated in micelles of dodecyl maltoside (DDM) and in phospholipid nanodiscs by monitoring the spatial positions of transmembrane helixes 6 and 7 at the cytoplasmic surface using site-directed spin labeling and double electron-electron resonance (DEER) spectroscopy. The photoactivated receptor in DDM was dominated by one conformation with weak pH dependence. In nanodiscs, however, an ensemble of pH-dependent conformational substates is obsd., even at pH 6.0 where the MIIbH+ form defined by proton uptake and optical spectroscopic methods is reported to be the sole species present in native disk membranes. In nanodiscs, the ensemble of substates in the photoactivated receptor spontaneously decayed to that characteristic of the inactive state with a lifetime of ∼16 min at 20°. Importantly, transducin binding to the activated receptor selected a subset of the ensemble in which multiple substates were apparently retained. The results indicated that in a native-like lipid environment rhodopsin activation was not analogous to a simple binary switch between 2 defined conformations, but the activated receptor was in equil. between multiple conformers that in principle could recognize different binding partners.
- 24Mary, S., Damian, M., Louet, M., Floquet, N., Fehrentz, J.-A., Marie, J., Martinez, J., and Baneres, J.-L. (2012) Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptor. Proc. Natl. Acad. Sci. U. S. A. 109, 8304– 8309, DOI: 10.1073/pnas.1119881109[Crossref], [PubMed], [CAS], Google Scholar24https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC38XosFahsr4%253D&md5=4e8027be4f2606fa695d6bb7e30bc247Ligands and signaling proteins govern the conformational landscape explored by a G protein-coupled receptorMary, Sophie; Damian, Marjorie; Louet, Maxime; Floquet, Nicolas; Fehrentz, Jean-Alain; Marie, Jacky; Martinez, Jean; Baneres, Jean-LouisProceedings of the National Academy of Sciences of the United States of America (2012), 109 (21), 8304-8309, S8304/1-S8304/23CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)The dynamic character of G protein-coupled receptors is essential to their function. However, the details of how ligands stabilize a particular conformation to selectively activate a signaling pathway and how signaling proteins affect this conformational repertoire remain unclear. Using a prototypical peptide-activated class A G protein-coupled receptor (GPCR), the ghrelin receptor, reconstituted as a monomer into lipid disks and labeled with a fluorescent conformational reporter, we demonstrate that ligand efficacy and functional selectivity are directly related to different receptor conformations. Of importance, our data bring direct evidence that distinct effector proteins affect the conformational landscape of the ghrelin receptor in different ways. Whereas G proteins affect the balance between active and inactive receptor substates in favor of the active state, agonist-induced arrestin recruitment is accompanied by a marked change in the structural features of the receptor that adopt a conformation different from that obsd. in the absence of arrestin. In contrast to G proteins and arrestins, μ-AP2 has no significant effect on the organization of the transmembrane core of the receptor. Such a modulation of a GPCR conformational landscape by pharmacol. distinct ligands and effectors provides insights into the structural bases that decisively affect ligand efficacy and subsequent biol. responses. This is also likely to have major implications for the design of drugs activating specific GPCR-assocd. signaling pathways.
- 25Gregorio, G. G., Masureel, M., Hilger, D., Terry, D. S., Juette, M., Zhao, H., Zhou, Z., Perez-Aguilar, J. M., Hauge, M., Mathiasen, S., Javitch, J. A., Weinstein, H., Kobilka, B. K., and Blanchard, S. C. (2017) Single-molecule analysis of ligand efficacy in β2AR-G-protein activation. Nature 547, 68– 73, DOI: 10.1038/nature22354[Crossref], [PubMed], [CAS], Google Scholar25https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXpt1Oqtb8%253D&md5=b7fc7f43e98bdf40611a7e7936b882afSingle-molecule analysis of ligand efficacy in β2AR-G-protein activationGregorio, G. Glenn; Masureel, Matthieu; Hilger, Daniel; Terry, Daniel S.; Juette, Manuel; Zhao, Hong; Zhou, Zhou; Perez-Aguilar, Jose Manuel; Hauge, Maria; Mathiasen, Signe; Javitch, Jonathan A.; Weinstein, Harel; Kobilka, Brian K.; Blanchard, Scott C.Nature (London, United Kingdom) (2017), 547 (7661), 68-73CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G-protein-coupled receptor (GPCR)-mediated signal transduction is central to human physiol. and disease intervention, yet the mol. mechanisms responsible for ligand-dependent signaling responses remain poorly understood. In class A GPCRs, receptor activation and G-protein coupling entail outward movements of transmembrane helix 6 (TM6). Here, using single-mol. fluorescence resonance energy transfer imaging, we examine TM6 movements in the β2 adrenergic receptor (β2AR) upon exposure to orthosteric ligands with different efficacies, in the absence and presence of the Gs heterotrimer. We show that partial and full agonists differentially affect TM6 motions to regulate the rate at which GDP-bound β2AR-Gs complexes are formed and the efficiency of nucleotide exchange leading to Gs activation. These data also reveal transient nucleotide-bound β2AR-Gs species that are distinct from known structures, and provide single-mol. perspectives on the allosteric link between ligand- and nucleotide-binding pockets that shed new light on the G-protein activation mechanism.
- 26Rasmussen, S., Choi, H., Fung, J., Pardon, E., Casarosa, P., Chae, P., DeVree, B., Rosenbaum, D., Thian, F., Kobilka, T. (2011) Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469, 175– 180, DOI: 10.1038/nature09648[Crossref], [PubMed], [CAS], Google Scholar26https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXkvFartA%253D%253D&md5=3a1d3bac6d92c9d54cf1ddd14d56ab8cStructure of a nanobody-stabilized active state of the β2 adrenoceptorRasmussen, Soren G. F.; Choi, Hee-Jung; Fung, Juan Jose; Pardon, Els; Casarosa, Paola; Chae, Pil Seok; DeVree, Brian T.; Rosenbaum, Daniel M.; Thian, Foon Sun; Kobilka, Tong Sun; Schnapp, Andreas; Konetzki, Ingo; Sunahara, Roger K.; Gellman, Samuel H.; Pautsch, Alexander; Steyaert, Jan; Weis, William I.; Kobilka, Brian K.Nature (London, United Kingdom) (2011), 469 (7329), 175-180CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviors in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behavior, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are assocd. with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those obsd. in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation.
- 27Schafer, C. T., Fay, J. F., Janz, J. M., and Farrens, D. L. (2016) Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor state. Proc. Natl. Acad. Sci. U. S. A. 113, 11961– 11966, DOI: 10.1073/pnas.1606347113[Crossref], [PubMed], [CAS], Google Scholar27https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC28Xhs1Srt7fI&md5=182075c0733d7899efe62eb9cac416a2Decay of an active GPCR: Conformational dynamics govern agonist rebinding and persistence of an active, yet empty, receptor stateSchafer, Christopher T.; Fay, Jonathan F.; Janz, Jay M.; Farrens, David L.Proceedings of the National Academy of Sciences of the United States of America (2016), 113 (42), 11961-11966CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Here, the authors describe 2 insights into the role of receptor conformational dynamics during agonist release (all-trans retinal; ATR) from the visual G protein-coupled receptor (GPCR), rhodopsin. First, the authors show that, after light activation, ATR can continually release and rebind to any receptor remaining in an active-like conformation. As with other GPCRs, the authors obsd. that this equil. could be shifted by either promoting the active-like population or increasing the agonist concn. Second, the authors found that during decay of the signaling state an active-like, yet empty, receptor conformation could transiently persist after retinal release, before the receptor ultimately collapses into an inactive conformation. The latter conclusion was based on time-resolved, site-directed fluorescence labeling expts. that showed a small, but reproducible, lag between retinal leaving the protein and the return of transmembrane helix 6 (TM6) to the inactive conformation, as detd. from tryptophan-induced quenching studies. Accelerating Schiff base hydrolysis and subsequent ATR dissocn., either by addn. of hydroxylamine or by introduction of mutations, further increased the time lag between ATR release and TM6 movement. These observations showed that rhodopsin can bind its agonist in equil. like a traditional GPCR, provided evidence that an active GPCR conformation can persist even after agonist release, and raised the possibility of targeting this key photoreceptor protein by traditional pharmaceutical-based treatments.
- 28Kato, H. E., Zhang, Y., Hu, H., Suomivuori, C.-M., Kadji, F. M. N., Aoki, J., Krishna Kumar, K., Fonseca, R., Hilger, D., Huang, W., Latorraca, N. R., Inoue, A., Dror, R. O., Kobilka, B. K., and Skiniotis, G. (2019) Conformational transitions of a neurotensin receptor 1-Gi1 complex. Nature DOI: 10.1038/s41586-019-1337-6 . [Epub ahead of print].
- 29Heck, M. and Hofmann, K. P. (2001) Maximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation: initial rate analysis based on a double displacement mechanism. J. Biol. Chem. 276, 10000– 10009, DOI: 10.1074/jbc.M009475200[Crossref], [PubMed], [CAS], Google Scholar29https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3MXis1OksLw%253D&md5=2987e5d6361cb577ab86e1969c595a5dMaximal rate and nucleotide dependence of rhodopsin-catalyzed transducin activation. Initial rate analysis based on a double displacement mechanismHeck, Martin; Hofmann, Klaus PeterJournal of Biological Chemistry (2001), 276 (13), 10000-10009CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Despite the growing structural information on receptors and G proteins, the information on affinities and kinetics of protein-protein and protein-nucleotide interactions is still not complete. In this study on photoactivated rhodopsin (R*) and the rod G protein, Gt, we have used kinetic light scattering, backed by direct biochem. assays, to follow G protein activation. Our protocol includes the following: (i) to measure initial rates on the background of rapid depletion of the GtGDP substrate; (ii) to titrate GtGDP, GTP, and GDP; and (iii) to apply a double displacement reaction scheme to describe the results. All data are simultaneously fitted by one and the same set of parameters. We obtain values of Km = 2200 Gt/μm2 for GtGDP and Km = 230 μM for GTP; dissocn. consts. are Kd = 530 Gt/μm2 for R*-GtGDP dissocn. and Kd = 270 μM for GDP release from R*GtGDP, once formed. Maximal catalytic rates per photoexcited rhodopsin are 600 Gt/s at 22° and 1300 Gt/s at 34°. The anal. provides a tool to allocate and quantify better the effects of chem. or mutational protein modifications to individual steps in signal transduction.
- 30Maeda, S., Qu, Q., Robertson, M. J., Skiniotis, G., and Kobilka, B. K. (2019) Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexes. Science 364, 552– 557, DOI: 10.1126/science.aaw5188[Crossref], [PubMed], [CAS], Google Scholar30https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXptFaks70%253D&md5=39d337fba0eff6fbb38bd8f2cbba3720Structures of the M1 and M2 muscarinic acetylcholine receptor/G-protein complexesMaeda, Shoji; Qu, Qianhui; Robertson, Michael J.; Skiniotis, Georgios; Kobilka, Brian K.Science (Washington, DC, United States) (2019), 364 (6440), 552-557CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science)Muscarinic acetylcholine receptors are G protein-coupled receptors that respond to acetylcholine and play important signaling roles in the nervous system. There are five muscarinic receptor subtypes (M1R to M5R), which, despite sharing a high degree of sequence identity in the transmembrane region, couple to different heterotrimeric GTP-binding proteins (G proteins) to transmit signals. M1R, M3R, and M5R couple to the Gq/11 family, whereas M2R and M4R couple to the Gi/o family. Here, we present and compare the cryo-electron microscopy structures of M1R in complex with G11 and M2R in complex with GoA. The M1R-G11 complex exhibits distinct features, including an extended transmembrane helix 5 and carboxyl-terminal receptor tail that interacts with G protein. Detailed anal. of these structures provides a framework for understanding the mol. determinants of G-protein coupling selectivity.
- 31Flock, T., Ravarani, C. N. J., Sun, D., Venkatakrishnan, A. J., Kayikci, M., Tate, C. G., Veprintsev, D. B., and Babu, M. M. (2015) Universal allosteric mechanism for Gα activation by GPCRs. Nature 524, 173– 179, DOI: 10.1038/nature14663[Crossref], [PubMed], [CAS], Google Scholar31https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2MXhtFyltLzF&md5=4eb6754be322f399caf2bbf831cec3ebUniversal allosteric mechanism for Gα activation by GPCRsFlock, Tilman; Ravarani, Charles N. J.; Sun, Dawei; Venkatakrishnan, A. J.; Kayikci, Melis; Tate, Christopher G.; Veprintsev, Dmitry B.; Babu, M. MadanNature (London, United Kingdom) (2015), 524 (7564), 173-179CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)G protein-coupled receptors (GPCRs) allosterically activate heterotrimeric G proteins and trigger GDP release. Given that there are ∼800 human GPCRs and 16 different Gα genes, this raises the question of whether a universal allosteric mechanism governs Gα activation. Here we show that different GPCRs interact with and activate Gα proteins through a highly conserved mechanism. Comparison of Gα with the small G protein Ras reveals how the evolution of short segments that undergo disorder-to-order transitions can decouple regions important for allosteric activation from receptor binding specificity. This might explain how the GPCR-Gα system diversified rapidly, while conserving the allosteric activation mechanism.
- 32Posner, B. A., Mixon, M. B., Wall, M. A., Sprang, S. R., and Gilman, A. G. (1998) The A326S mutant of Gialpha1 as an approximation of the receptor bound state. J. Biol. Chem. 273, 21752– 21758, DOI: 10.1074/jbc.273.34.21752[Crossref], [PubMed], [CAS], Google Scholar32https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaK1cXlsFOqs7c%253D&md5=58f0a3547839f3f02b7b6023bf63da1cThe A326S mutant of Giα1 as an approximation of the receptor-bound statePosner, Bruce A.; Mixon, Mark B.; Wall, Mark A.; Sprang, Stephen R.; Gilman, Alfred G.Journal of Biological Chemistry (1998), 273 (34), 21752-21758CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)Agonist-bound heptahelical receptors activate heterotrimeric G proteins by catalyzing exchange of GDP for GTP on their α subunits. In search of an approxn. of the receptor-α subunit complex, we have considered the properties of A326S Giα1, a mutation discovered originally in Gsα (Iiri, T., Herzmark, P., Nakamoto, J. M., Van Dop, C., and Bourne, H. R. (1994) Nature 371, 164-168) that mimics the effect of receptor on nucleotide exchange. The mutation accelerates dissocn. of GDP from the αi1β1γ2 heterotrimer by 250-fold. Nevertheless, affinity of mutant Giα1 for GTPγS is high in the presence of Mg2+, and the mutation has no effect on the intrinsic GTPase activity of the α subunit. The mutation also uncouples two activities of βγ: stabilization of the GDP-bound α subunit (which is retained) and retardation of GDP dissocn. from the heterotrimer (which is lost). For wild-type and mutant Giα1, βγ prevents irreversible inactivation of the α subunit at 30°. However, the mutation accelerates irreversible inactivation of α at 37° despite the presence of βγ. Structurally, the mutation weakens affinity for GTPγS by steric crowding: a 2-fold increase in the no. of close contacts between the protein and the purine ring of the nucleotide. By contrast, we observe no differences in structure at the GDP binding site between wild-type heterotrimers and those contg. A326S Giα1. However, the GDP binding site is only partially occupied in crystals of G protein heterotrimers contg. A326S Giα1. In contrast to original speculations about the structural correlates of receptor-catalyzed nucleotide exchange, rapid dissocn. of GDP can be obsd. in the absence of substantial structural alteration of a Gα subunit in the GDP-bound state.
- 33Brandt, D. R. and Ross, E. M. (1985) GTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediates. J. Biol. Chem. 260, 266– 272[Crossref], [PubMed], [CAS], Google Scholar33https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2MXptF2ltw%253D%253D&md5=41ebfc79dbf08543f4f6e5fda12e4a4aGTPase activity of the stimulatory GTP-binding regulatory protein of adenylate cyclase, Gs. Accumulation and turnover of enzyme-nucleotide intermediatesBrandt, Douglas R.; Ross, Elliott M.Journal of Biological Chemistry (1985), 260 (1), 266-72CODEN: JBCHA3; ISSN:0021-9258.The GTPase activity of the stimulatory guanine nucleotide-binding regulatory protein (Gs) of hormone-sensitive adenylate cyclase was investigated using purified rabbit hepatic Gs and either [α-32P]- or [γ-32P]GTP as substrate. The binding of [35S]guanosine 5'-O-(thiotriphosphate) (GTPγS) was used to quant. the total concn. of Gs. GTPase activity was a saturable function of the concn. of GTP, with a Km of 0.3 μM. MgCl2 monotonically increased the activity. The max. obsd. turnover no. was ∼ 1.5 min-1. During steady-state hydrolysis, 20-40% of total Gs could be trapped as a Gs-GDP complex and 1-2% could be trapped as Gs-GTP. The hydrolysis of Gs-GTP to Gs-GDP occurred with t1/2 of ≤5 s at 30° and t1/2 ∼1 min at 0°. Hydrolysis of Gs-GTP was inhibited by 1.0 mM EDTA in the absence of added Mg2+. The rate of formation of Gs-GDP and the initial GTPase rate varied in parallel as functions of the concns. of either GTP or MgCl2 (>0.1 mM Mg2+). The ratio of the rate of accumulation of Gs-GDP to the GTPase rate was const. at 0.3-0.4. The rate of dissocn. of assayable Gs-GDP was biphasic. The initial phase accounted for 60-80% of total assayable Gs-GDP and was characterized by a t1/2 of ∼ 1 min. Lubrol 12A9 potently inhibited the GTPase reaction and the dissocn. of Gs-GDP in parallel, and inhibition of product release may account for the inhibition of steady-state hydrolysis. The β and γ subunits of Gs markedly inhibited the dissocn. of GDP from Gs, in contrast to their ability to stimulate the dissocn. of GTPγS. GDP, GTPγS, and guanyl-5'-yl imidodiphosphate [Gpp(NH)p] competitively inhibited the accumulation of Gs-GDP. GTPγS and Gpp(NH)p inhibited the GTPase reaction noncompetitively, GDP displayed mixed inhibition, and inorg. phosphate did not inhibit. Thus 2 specific mechanistic pathways for the overall GTPase reaction may coexist.
- 34Higashijima, T., Ferguson, K. M., Sternweis, P. C., Smigel, M. D., and Gilman, A. G. (1987) Effects of Mg2+ and the beta gamma-subunit complex on the interactions of guanine nucleotides with G proteins. J. Biol. Chem. 262, 762– 766[Crossref], [PubMed], [CAS], Google Scholar34https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlartg%253D%253D&md5=c7868e70f8e76b0da863c19678340ff7Effects of magnesium and the βγ-subunit complex on the interactions of guanine nucleotides with G proteinsHigashijima, Tsutomu; Ferguson, Kenneth M.; Sternweis, Paul C.; Smigel, Murray D.; Gilman, Alfred G.Journal of Biological Chemistry (1987), 262 (2), 762-6CODEN: JBCHA3; ISSN:0021-9258.Mg2+ interacts with the α subunits of guanine nucleotide-binding regulatory proteins (G proteins) in the presence of guanosine-5'-[γ-thio]triphosphate (GTPγS) to form a highly fluorescent complex from which nucleotide dissocs. very slowly. The apparent dissocn. const. for interaction of Gα·GTPγS with Mg2+ is ∼5 nM, similar to the Km for G protein GTPase activity. Gβγ increases the rate of dissocn. of GTPγS from Gα·GTPγS or Gα·GTPγS·Mg2+ at low Mg2+ concns. When the concn. of Mg2+ is >1 mM, Gβγ dissocs. from Gβγ·Gα·GTPγS·Mg2+. Compared with the dramatic effect of Mg2+ on binding of GTPγS to Gα, the metal has relatively little effect on the binding of GDP. However, Bβγ increases the affinity of Gα for GDP by >100-fold. High concns. of Mg2+ promote the dissocn. of GDP from Gβγ·Gα·GDP, apparently without causing subunit dissocn. The steady-state rate of GTP hydrolysis is strictly correlated with the rate of dissocn. of GDP from Gα under all conditions examd. Thus, there are ≥2 sites for interaction of Mg2+ with G protein-nucleotide complexes. Furthermore, binding of Gβγ and GTPγS to Gα is neg. cooperative, whereas the binding interaction between Gβγ and GDP is strongly pos.
- 35Sun, X., Singh, S., Blumer, K., and Bowman, G. R. (2018) Simulation of spontaneous G protein activation reveals a new intermediate driving GDP unbinding. eLife 7, e38465, DOI: 10.7554/eLife.38465[Crossref], [PubMed], [CAS], Google Scholar35https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXitlyqsr7E&md5=9fb2812a94176f80c028c0b26751375cSimulation of spontaneous G protein activation reveals a new intermediate driving GDP unbindingSun, Xianqiang; Singh, Sukrit; Blumer, Kendall J.; Bowman, Gregory R.eLife (2018), 7 (), e38465/1-e38465/24CODEN: ELIFA8; ISSN:2050-084X. (eLife Sciences Publications Ltd.)Activation of heterotrimeric G proteins is a key step in many signaling cascades. However, a complete mechanism for this process, which requires allosteric communication between binding sites that are ∼30 Å apart, remains elusive. We construct an atomically detailed model of G protein activation by combining three powerful computational methods: metadynamics, Markov state models (MSMs), and CARDS anal. of correlated motions. We uncover a mechanism that is consistent with a wide variety of structural and biochem. data. Surprisingly, the rate-limiting step for GDP release correlates with tilting rather than translation of the GPCR-binding helix 5. b- Strands 1 - 3 and helix 1 emerge as hubs in the allosteric network that links conformational changes in the GPCR-binding site to disordering of the distal nucleotide-binding site and consequent GDP release. Our approach and insights provide foundations for understanding disease-implicated G protein mutants, illuminating slow events in allosteric networks, and examg. unbinding processes with slow off-rates.
- 36Higashijima, T., Ferguson, K. M., Smigel, M. D., and Gilman, A. G. (1987) The effect of GTP and Mg2+ on the GTPase activity and the fluorescent properties of Go. J. Biol. Chem. 262, 757– 761[Crossref], [PubMed], [CAS], Google Scholar36https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL2sXmtlarsQ%253D%253D&md5=76ae6d8869065a77e884a6369f03e142The effect of GTP and magnesium on the GTPase activity and the fluorescent properties of GoHigashijima, Tsutomu; Ferguson, Kenneth M.; Smigel, Murray D.; Gilman, Alfred G.Journal of Biological Chemistry (1987), 262 (2), 757-61CODEN: JBCHA3; ISSN:0021-9258.GTP causes an increase in the fluorescence of Go, a G protein from bovine brain. When Mg2+ is also present, the increase in fluorescence is transient, and the rate of decline in the intensity of the fluorescence is the same as the rate of GTP hydrolysis by the protein. The steady-state rate of hydrolysis of GTP by Go (0.3-0.4/min) is slower than the catalytic rate of the protein (2/min), because the rate-limiting step in the reaction is the release of GDP.
- 37Du, Y., Duc, N. M., Rasmussen, S. G. F., Hilger, D., Kubiak, X., Wang, L., Bohon, J., Kim, H. R., Wegrecki, M., Asuru, A., Jeong, K. M., Lee, J., Chance, M. R., Lodowski, D. T., Kobilka, B. K., and Chung, K. Y. (2019) Assembly of a GPCR-G Protein Complex. Cell 177, 1232– 1242, DOI: 10.1016/j.cell.2019.04.022[Crossref], [PubMed], [CAS], Google Scholar37https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVOqtbk%253D&md5=7e1c310efcda7dcc2275955887273d09Assembly of a GPCR-G Protein ComplexDu, Yang; Duc, Nguyen Minh; Rasmussen, Soeren G. F.; Hilger, Daniel; Kubiak, Xavier; Wang, Liwen; Bohon, Jennifer; Kim, Hee Ryung; Wegrecki, Marcin; Asuru, Awuri; Jeong, Kyung Min; Lee, Jeongmi; Chance, Mark R.; Lodowski, David T.; Kobilka, Brian K.; Chung, Ka YoungCell (Cambridge, MA, United States) (2019), 177 (5), 1232-1242.e11CODEN: CELLB5; ISSN:0092-8674. (Cell Press)The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallog. and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are obsd., there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is detd. by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes obsd. in crystal and cryo-EM structures.
- 38Roberts, D. J., Lin, H., and Strange, P. G. (2004) Mechanisms of agonist action at D2 dopamine receptors. Mol. Pharmacol. 66, 1573– 1579, DOI: 10.1124/mol.104.004077[Crossref], [PubMed], [CAS], Google Scholar38https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD2cXhtVOit7vJ&md5=6954bf6bdef1dc313e0d8b24464095eaMechanisms of agonist action at D2 dopamine receptorsRoberts, David J.; Lin, Hong; Strange, Philip G.Molecular Pharmacology (2004), 66 (6), 1573-1579CODEN: MOPMA3; ISSN:0026-895X. (American Society for Pharmacology and Experimental Therapeutics)In this study, we investigated the biochem. mechanisms of agonist action at the G protein-coupled D2 dopamine receptor expressed in Chinese hamster ovary cells. Stimulation of guanosine 5'-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding by full and partial agonists was detd. at different concns. of [35S]GTPγS (0.1 and 10 nM) and in the presence of different concns. of GDP. At both concns. of [35S]GTPγS, increasing GDP decreased the [35S]GTPγS binding obsd. with maximally stimulating concns. of agonist, with partial agonists exhibiting greater sensitivity to the effects of GDP than full agonists. The relative efficacy of partial agonists was greater at the lower GDP concns. Concn.-response expts. were performed for a range of agonists at the two [35S]GTPγS concns. and with different concns. of GDP. At 0.1 nM [35S]GTPγS, the potency of both full and partial agonists was dependent on the GDP concn. in the assays. At 10 nM [35S]GTPγS, the potency of full agonists exhibited a greater dependence on the GDP concn., whereas the potency of partial agonists was virtually independent of GDP. We concluded that at the lower [35S]GTPγS concn., the rate-detg. step in G protein activation is the binding of [35S]GTPγS to the G protein. At the higher [35S]GTPγS concn., for full agonists, [35S]GTPγS binding remains the slowest step, whereas for partial agonists, another (GDP-independent) step, probably ternary complex breakdown, becomes rate-detg.
- 39Grant, B. J., McCammon, J. A., and Gorfe, A. A. (2010) Conformational selection in G-proteins: lessons from Ras and Rho. Biophys. J. 99, L87– 9, DOI: 10.1016/j.bpj.2010.10.020[Crossref], [PubMed], [CAS], Google Scholar39https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXhsV2js7fJ&md5=7fe80e3aab3fa9e57e7bb5e3c51860eaConformational selection in G-proteins: Lessons from Ras and RhoGrant, Barry J.; McCammon, J. Andrew; Gorfe, Alemayehu A.Biophysical Journal (2010), 99 (11), L87-L89CODEN: BIOJAU; ISSN:0006-3495. (Cell Press)The induced fit model has traditionally been invoked to describe the activating conformational change of monomeric G-proteins, such as Ras and Rho. With this scheme, the presence or absence of the γ-phosphate of GTP leads to an instantaneous switch in conformation. Here, the authors describe atomistic mol. simulations that demonstrate that both Ras and Rho superfamily members harbor an intrinsic susceptibility to sample multiple conformational states in the absence of nucleotide ligand. By comparing the distribution of conformers in the presence and absence of nucleotide, the authors show that conformational selection is the dominant mechanism by which Ras and Rho undergo nucleotide-dependent conformational changes. Furthermore, the pattern of correlated motions revealed by these simulations predicts a preserved allosteric coupling of the nucleotide-binding site with the membrane interacting C-terminus in both Rho and Ras.
- 40Flock, T., Hauser, A. S., Lund, N., Gloriam, D. E., Balaji, S., and Babu, M. M. (2017) Selectivity determinants of GPCR-G-protein binding. Nature 545, 317– 322, DOI: 10.1038/nature22070[Crossref], [PubMed], [CAS], Google Scholar40https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXnsVeis74%253D&md5=947db386e0e0f74b09465a99d880c518Selectivity determinants of GPCR-G-protein bindingFlock, Tilman; Hauser, Alexander S.; Lund, Nadia; Gloriam, David E.; Balaji, Santhanam; Babu, M. MadanNature (London, United Kingdom) (2017), 545 (7654), 317-322CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)The selective coupling of G-protein-coupled receptors (GPCRs) to specific G proteins is crit. to trigger the appropriate physiol. response. However, the determinants of selective binding have remained elusive. Here, we reveal the existence of a selectivity barcode (i.e., patterns of amino acids) on each of the 16 human G proteins that is recognized by distinct regions on the ∼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 nonidentical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-detg. residues. These findings lay the foundation for understanding the mol. basis of coupling selectivity within individual receptors and G proteins.
- 41Gupte, T. M., Malik, R. U., Sommese, R. F., Ritt, M., and Sivaramakrishnan, S. (2017) Priming GPCR signaling through the synergistic effect of two G proteins. Proc. Natl. Acad. Sci. U. S. A. 114, 3756– 3761, DOI: 10.1073/pnas.1617232114[Crossref], [PubMed], [CAS], Google Scholar41https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXks1Smtrk%253D&md5=d3d4c469231ebfef8e908e28199c0b8dPriming GPCR signaling through the synergistic effect of two G proteinsGupte, Tejas M.; Malik, Rabia U.; Sommese, Ruth F.; Ritt, Michael; Sivaramakrishnan, SivarajProceedings of the National Academy of Sciences of the United States of America (2017), 114 (14), 3756-3761CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimol. event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming.". Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggesting an altered GPCR conformation as the underlying basis for GPCR priming. We propose three parallel mechanisms involving (i) sequential G-protein interactions at the cognate site, (ii) G-protein interactions at distinct allosteric and cognate sites on the GPCR, and (iii) asym. GPCR dimers. GPCR priming suggests another layer of regulation in the classic GPCR ternary-complex model, with broad implications for the multiplicity inherent in signaling networks.