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Biochem. J. (2013) 450, 443–457 (Printed in Great Britain) doi:10.1042/BJ20121644 443

REVIEW ARTICLEG-protein-coupled receptor structure, ligand binding and activation asstudied by solid-state NMR spectroscopyXiaoyan DING*, Xin ZHAO*†1 and Anthony WATTS‡1

*Shanghai Key Laboratory of Magnetic Resonance, Department of Physics, East China Normal University, Shanghai 200062, P.R. China, †Center for Laser and ComputationalBiophysics, State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062, P.R. China, and ‡Biomembrane Structure Unit, Department ofBiochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

GPCRs (G-protein-coupled receptors) are versatile signallingmolecules at the cell surface and make up the largest and mostdiverse family of membrane receptors in the human genome. Theyconvert a large variety of extracellular stimuli into intracellularresponses through the activation of heterotrimeric G-proteins,which make them key regulatory elements in a broad rangeof normal and pathological processes, and are therefore one ofthe most important targets for pharmaceutical drug discovery.Knowledge of a GPCR structure enables us to gain a mechanisticinsight into its function and dynamics, and further aid rationaldrug design. Despite intensive research carried out over the lastthree decades, resolving the structural basis of GPCR functionis still a major activity. The crystal structures obtained in thelast 5 years provide the first opportunity to understand howprotein structure dictates the unique functional properties of thesecomplex signalling molecules. However, owing to the intrinsichydrophobicity, flexibility and instability of membrane proteins, itis still a challenge to crystallize GPCRs, and, when this is possible,it is no longer in its native membrane environment and no longer

without modification. Furthermore, the conformational changeof the transmembrane α-helices associated with the structureactivation increases the difficulty of capturing the activation stateof a GPCR to a higher resolution by X-ray crystallography. On theother hand, solid-state NMR may offer a unique opportunity tostudy membrane protein structure, ligand binding and activationat atomic resolution in the native membrane environment, as wellas described functionally significant dynamics. In the presentreview, we discuss some recent achievements of solid-state NMRfor understanding GPCRs, the largest mammalian proteome at∼1% of the total expressed proteins. Structural information,details of determination, details of ligand conformations and theconsequences of ligand binding to initiate activation can all beexplored with solid-state NMR.

Key words: conformational dynamics, G-protein-coupledreceptor (GPCR), ligand binding and structural activation,solid-state NMR, structure determination.

INTRODUCTION

GPCRs (G-protein-coupled receptors) are versatile signallingmolecules at the cell surface and make up the largest and mostdiverse family of membrane receptors in the human genome,converting a large variety of extracellular stimuli, includinglight, odorants, ions, lipids, catecholamines, neuropeptides andlarge glycoprotein hormones into intracellular response throughthe activation of heterotrimeric G-proteins [1], which makethem the key regulatory elements in a broad range of normaland pathological processes and the most important targets forpharmaceutical drug discovery [2–4]. Analysis of the humangenome revealed at least 799 GPCRs in humans, comprisingthe largest family of such receptors within most mammals [5,6].GPCRs are widely classified into five main families on the basisof their sequence similarity: the rhodopsin family (701 members),the adhesion family (24 members), the frizzled/taste family (24members), the glutamate family (15 members) and the secretinfamily (15 members), forming the GRAFS classification system[5]. The rhodopsin family is by far the largest and most diverse ofthese families, forming four main groups with 13 sub-branches,and the members are characterized by conserved sequence

motifs and are thought to share the similar activation mechanisms.GPCRs share a common 7TM (seven-transmembrane) α-helixarchitecture and couple to G-proteins [7]. The TM α-helicesare connected by alternating three extracellular and cytoplasmicloops (EL1–EL3 and CL1–CL3), with the NT (N-terminus)extracellular and the CT (C-terminus) intracellular, arrangedin an anticlockwise fashion as viewed from the extracellularsurface. The crystal structures of rhodopsin [8–10], β1AR (β1-adrenergic receptor) and β2AR (β2-adrenergic receptor) [11,12],A2a adenosine receptor [13], CXCR4 chemokine receptor [14],dopamine D3 receptor [15], the human histamine H1 receptor[16], the M2 and M3 muscarinic acetylcholine receptors [17,18],the human κ-opioid receptor [19], the nociceptin/orphanin FQreceptor [20], the μ-opioid receptor [21] and a very recentstructure of the agonist-bound neurotensin receptor [22] at highresolution have confirmed this structural topology. Binding ofa stimulus, the so-called ‘first messenger’, to the extracellularor TM domains of a GPCR triggers conformational changes inthe 7TM structure that are transmitted through the intracellularreceptor domains to promote coupling between the receptor andits cognate heterotrimeric G-proteins. The receptor stimulatesG-protein activation by catalysing the exchange of GTP for

Abbreviations used: β1AR, β1-adrenergic receptor; β2AR, β2-adrenergic receptor; CL, cytoplasmic loop; CP, cross-polarization; CT, C-terminus; DARR,dipolar-assisted rotational resonance; DNP, dynamic nuclear polarization; DQF, double quantum filtration; EL, extracellular loop; GPCR, G-protein-coupledreceptor; HEK, human embryonic kidney; HH, Hartmann–Hahn; MAS, magic-angle spinning; NT, N-terminus; PDSD, proton-driven spin diffusion; PISEMA,Polarization Inversion Spin Exchange at the Magic Angle; SAR, structure–activity relationship; SSNMR, solid-state NMR; 7TM, seven-transmembrane.

1 Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).

c© The Authors Journal compilation c© 2013 Biochemical Society

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444 X. Y. Ding, X. Zhao and A. Watts

GDP on the Gα subunit and dissociation of the GTP-bound Gα

subunit from the Gβγ subunit heterodimer. Once dissociated, freeGα-GTP and Gβγ subunits regulate the activity of enzymaticeffectors, such as adenylate cyclases, PLC (phospholipase C)isoforms and ion channels, to generate small molecules, the‘second messengers’. The second messengers, in turn, controlthe activity of protein kinases that regulate key enzymes involvedin intermediate metabolism. Hydrolysis of GTP to GDP withinGα and subsequent reassociation of Gα-GDP and Gβγ completesthe G-protein cycle [23–26].

GPCRs share not only a similar overall structural topology,but also a similar activation mechanism [1,27]. Despite thefact that these receptors are activated by various agonistswhich are different in chemical nature, many biochemical andbiophysical studies have indicated that the receptor activationis associated with a relatively large overall rearrangement ofthe TMs [28,29], as described by the global toggle switchactivation model [30,31]. By which, the TM VI undergoes a‘vertical’ see-saw movement around a pivot corresponding to thehighly conserved proline residue in the middle of the membraneduring activation, resulting in an inward rotation around themain ligand-binding pocket on the extracellular side and anoutward rotation on the intracellular side to open a space forG-protein binding. This global activation mechanism is integratedby several molecular microswitches, defined as residues that havebeen highly conserved during evolution and that switch betweensubstantially different conformations in the active compared withthe inactive state of the Class A GPCRs, as illustrated by the Rhomodel [27,32,33]. For example, the Arg135 (3.50) (denoted by theBallesteros–Weinstein numbering index [34]) switch of the highlyconserved (E/D)RY motif functions as a microswitch throughinterchanging conformation from inactive by interacting withAsp134 (3.49) to active by interacting with Tyr223 (5.58) and theG-protein. The Trp265 (6.48) rotamer switch of the CWXP motif inTM VI functions as a microswitch through interchanging betweenan inactive rotamer conformation in which the indole side chaininteracts with a structural water molecule of the hydrogen-bondnetwork, and an active rotamer conformation in which the indoleinteracts with Phe208 (5.43) at the bottom of the binding pocket.The Tyr306 (7.53) rotamer switch of the NPXXY motif in TMVII interchanges between an inactive rotamer conformation byinteracting with Phe313 and an active rotamer conformationby participating in a hydrophobic cluster between the active con-formations of TM VI and TM VII [33,35], as shown in Figure 1.

Despite intensive research having been carried out over thelast three decades, more needs to be known about the structuralbasis of GPCR function. The crystal structures obtained inthe last 5 years provide the first opportunity to understandhow protein structure dictates the unique functional propertiesof these complex signalling molecules. Furthermore, owingto intrinsic hydrophobicity, flexibility and thermoinstability ofmembrane proteins, far fewer structures have been solved byX-ray crystallography for membrane proteins, especiallythe GPCRs, than for soluble proteins [36,37]. Moreover, theconformational change of the TM α-helices associated withthe structure activation increases the difficulty of capturing theactivation state of a membrane protein to a higher resolutionby X-ray crystallography [38,39]. On the contrary, SSNMR(solid-state NMR) has no requirement for the sample form andmolecular mass in principle, and is a suitable technique to studyGPCR structure, ligand binding, dynamics and the conformationalchanges associated with structural activations at the atomic levelin the native membrane environments.

Over the last few years, SSNMR has made tremendousprogress showing its capability of determining membrane protein

Figure 1 Schematic overview of GPCR global toggle switch model coupledwith microswitches and extra- and intracellular ligand-binding pockets

Red arrows indicate the major ‘vertical’ see-saw movement of TM VI occurring during the globaltoggle switch activation inward into the main ligand-binding pocket and outward at theintracellular end to open for G-protein binding. The three main microswitches are indicatedin both their inactive form and in their proposed active form (blue): the Trp265 (6.48) rotamerswitch at the bottom of the binding pocket, and the Tyr306 (7.53) rotamer switch and the Arg135

(3.50) switch, which are both located at the cytoplasmic face of the receptor. Residues involved inthe binding of the C-terminal helical segment of the Gα subunit are indicated in green, whichin the folded protein will line the binding cavity that is opened up in the active receptorconformation. Reprinted from Trends in Pharmacological Sciences, 30/5, Rie Nygaard, ThomasM. Frimurer, Birgitte Holst, Mette M. Rosenkilde, Thue W. Schwartz, Ligand binding andmicro-switches in 7TM receptor structures, 249–259, c© 2009, with permission from Elsevier.

structure, ligand binding and protein dynamic conformation ona variety of timescales at atomic resolution. Many membraneproteins have been investigated by MAS (magic-angle spinning)SSNMR and static-oriented SSNMR, including activation,inhibition and dynamics of the K+ channel KcsA–Kv1.3 [40–45];ligand conformation, photointermediates, structural activationand dynamics of rhodopsin [46–63]; the ligand binding andconstitutive dimerization of neurotensin receptor 1 [64,65];the protonation switch mechanism of the human histamineH1 receptor [66]; the influenza M2 proton channel structure,function, ligand binding and membrane mediation [67–73]; andthe structure, dynamics and lipid interaction of the retinylideneproteins from bacteria, including bacteriorhodopsin, sensoryrhodopsin, halorhodopsin and proteorhodopsin [74–85]. Someother membrane proteins, such as membrane-embedded enzymes[86–88], histidine kinases [89], ABC (ATP-binding cassette)transporters [90] and bacterial outer membrane proteins [91,92],have also been investigated through multidimensional correlationexperiments in MAS SSNMR.

In the present review, we briefly discuss some of the recentprogress in studying GPCRs by using MAS or static SSNMRfrom a structural biology point of view. For a complete overviewof the achievements in this field, please refer to some other reviews[32,93–108].

GENERAL TECHNIQUES USED IN SOLID-STATE NMR FORMEMBRANE PROTEINS

Unlike solution-state NMR, the resolution and sensitivity ofSSNMR are dependent on orientationally dependent anisotropic


GPCR and solid-state NMR 445

spin interactions, such as chemical shift anisotropy, homonuclearand heteronuclear dipole–dipole couplings or quadrupolarcouplings. These interactions generally cannot be averaged outby the molecular tumbling motions in solids, presenting a verybroad line-shape with poor sensitivity of SSNMR spectra. Ifthe sample rotor is spun about an axis with a tilt angle of54.74◦, the magic angle, with respect to the static field B0, thoseanisotropic spin interactions will be largely averaged out by thespatial rotation [109]. As the spinning frequency increases,the suppression process will be much more efficient and complete,which will lead to a solution-state-like spectrum.

Similar to the INEPT (Insensitive Nuclei EnhancementPolarization Transfer) heteronuclear J-coupling transfer schemein solution-state NMR [110], the sensitivity of the rare spin specieswith low magnetogyric ratio, such as 13C and 15N can be enhancedby transferring magnetization from abundant spin species withhigh magnetogyric ratio, such as 1H through the heteronucleardipolar couplings via CP (cross-polarization) scheme in solids[111]. Magnetization transfer is achieved when the strengthsof the two fields match the HH (Hartmann–Hahn) condition∣∣γI B I

r f

∣∣ = ∣

∣γS BSr f

∣∣, where γ I and γ S are the magnetogyric ratios of

the I-spins and S-spins respectively [112]. Ramping or adiabatic-pulsing one of the rf fields can improve the reliability andreproducibility of HH-CP, especially in fast MAS experiments.

The couplings between protons and the rare spins with lowγ , such as 13C and 15N often complicate observation of the rarespins, therefore it is usually necessary to remove those strongheteronuclear dipolar couplings by using strong rf irradiationon proton spins, i.e. so-called high-power proton decoupling. CW(continuous wave) decoupling is a standard scheme for decouplingof the heteronuclear dipolar spin interactions at static or slow MASconditions [113], TPPM, Spinal64 and other more sophisticatedpulse sequences are used for fast MAS in order to achieve a betterdecoupling efficiency [114,115].

For sequential assignment of protein structures in SSNMR, fastMAS is essential to gain high resolution and sensitivity. However,all anisotropic spin interactions which can be used to extractmolecular geometry information are averaged out by MAS. Inorder to restore the anisotropic spin interactions in the presenceof MAS, recoupling techniques were developed to selectivelyretrieve the anisotropic spin interactions for rotating solids[116,117]. Generally, there are two approaches to reintroduceanisotropic dipolar interactions, either mechanically, where therecoupling is achieved through rotational resonance, or by rfpulse-driven methods where the recoupling is achieved byapplying rf pulse trains. By applying recoupling sequences,certain anisotropic interaction terms can be retrieved in thepresence of MAS [118].

Combining MAS with cross-polarization, high-power protondecoupling, selective recoupling and multi-dimensional exper-imental scheme with isotopic labelling, GPCRs structure anddynamic conformation associated with activation can be revealedthrough the short- and long-range semi-quantitative distancerestraints from multi-dimensional homonuclear and heteronuclearcorrelation experiments to a high resolution and signal-to-noiseratio in SSNMR for GPCR structure and function studies [48–57,62–65,119–122].

EXPRESSION SYSTEMS AND ISOTOPE LABELLING STRATEGIES

GPCRs are generally expressed at low levels in their endogenousenvironment, but milligram level of samples are essential forSSNMR to carry out various multi-dimensional experimentalstudies on GPCR structure and function, so the ability to express

and purify enough amount of functional receptors is a demandingand challenging task to SSNMR spectroscopists [123]. GPCRshave been expressed in Escherichia coli, yeast, insect andmammalian cells and cell-free systems for various studies [124–138], and the advantages and disadvantages of each system forGPCR expression are summarized in Table 1. In the presentreview, we briefly mention some of those systems.

Low cost, ease of culture, optimized strains and lack of post-translational modifications, higher scalability and very flexibleisotope labelling strategies are the advantages of expressingGPCRs in E. coli. However, glycosylation and palmitoylationhave been reported to be needed for proper folding and functionof GPCRs, such as ligand binding in ligand-activated GPCRs,and lack of some eukaryotic membrane components, such ascholesterol can also affect receptor activity [128,134,138]. Themain reason is probably that E. coli is a prokaryote, whereas allligand-binding GPCRs identified are eukaryotic [97].

Mammalian cells provide numerous advantages for GPCRexpression since they contain the necessary co-receptors andmembrane composition to provide the highest level of post-translational modifications for the correct folding of functionalGPCRs, and the proteins can be expressed transiently or stably viaa selective process. HEK (human embryonic kidney)-293S cellshave been used to express several different GPCRs successfullyin media containing specifically labelled amino acids [49–55,130,131,139–142]. Although mammalian expression systemsfor GPCRs are relatively costly and time-consuming, if they arescalable, they have the promise to provide suitable concentrationsof active correctly modified receptors for biophysical studies [97].

Insect cells are used in conjunction with a baculovirus whichallows transfection of DNA encoding the GPCR of interest into thecell [143]. This eukaryotic expression system offers the advantagethat it can be readily adapted to high-density suspension culture forlarge-scale expression and allows many of the post-translationalmodifications present in mammalian systems. Insect cells suchas the commonly used Sf9 cells from Spodoptera frugiperdahave numerous advantages and have been used for expressingisotope-labelled GPCRs [144,145], but also have some importantdisadvantages for GPCR expression, as summarized in Table 1.

Cell-free expression offers a unique tool in the preparation ofprotein samples for biophysical studies. The near total controlover labelling strategies allowed by this system, along withother advantages makes this potentially one of the most significantadvances in protein production in recent years [136,137]. A keycomponent of cell-free expression systems is the cell lysate usedas the basis for the reaction mixture. Cell-free expression systemshave the highest flexibility of isotopic labelling. It can incorporatea single 13C- and 15N-labelled amino acid or one amino acid with13C and another with 15N to allow assignment of pairs of aminoacids. It is also possible to attach a single labelled amino acid ata specific position by using engineered tRNA molecules [146] inorder to reduce dipolar truncation and cross-relaxation broadeningcaused by the directly bound pair of labels. Although most of thesetechniques have not yet been applied to GPCRs generated by cell-free expression, it is clear that this system has significant potentialfor NMR analysis of such receptors.

Isotopic labelling plays a very important role in molecularstructure determination in SSNMR. It can not only enhancethe spectral sensitivity and improve the spectral resolution, butalso helps with the resonance assignments of NMR spectraand tackles the specific problem of the structure and dynamicsof proteins through the designed labelling scheme. There arethree main approaches to isotopically label a protein: specific,selective and uniform labelling. Specific labelling normally refersto incorporation of one or several 13C,15N-labelled amino acids



Table 1 Comparison of GPCR expression systems for NMR studies

Table adapted from Tapaneeyakorn, S., Goddard, A.D., Oates, J., Willis, C.L. and Watts, A. (2011) Solution- and solid-state NMR studies of GPCRs and their ligands. Biochim. Biophys. Acta 1808,1462–1475 with permission.

Expression system Advantages Disadvantages

E. coli Inexpensive Lack of post-translational modification, e.g. glycosylation is required for someGPCR–ligand interactions

Ease of culture Lack of some eukaryotic membrane components including cholesterol can affectreceptor activity

Genetic flexibility High-level expression may result in formation of inclusion bodies; limited successin refolding GPCRs

Strains optimized for protein expression Different codon usage (overcome by expression of rare tRNAs)Very flexible isotope labelling strategies Low success rate of active GPCR expressionHigh scalability, although there is not necessarily a linear relationship between

scale and yieldYeast Inexpensive Endogenous GPCRs and G-proteins which may interfere with expression and

purificationEase of culture Different membrane compositionGenetic flexibility Relatively thick cell wall may impede purificationGood scalability Labelling strategies more limited than some systemsCellular compartmentalizationEukaryotic post-translational modificationsCan co-express accessory proteins

Baculovirus/insect cells Eukaryotic; nearly all post-translational modifications are identical with those ofmammalian cells

Glycosylation is different from that of animal cells

Most GPCRs expressed are active Membrane is higher in unsaturated fatty acids and lower in cholesterol which canbe key in GPCR activity

Insect cells are semi-adherent and also grow in suspension allowing goodscalability, e.g. fermenter growths

Requires high virus titre

Commercialization has reduced costs Cultures are only stable for ∼1 month owing to accumulation of defective virusparticles

Limited labelling strategies availableMammalian cells Native cellular environment Relatively expensive

Correct trafficking and folding Possible complications from endogenous receptors and signalling componentsCorrect membrane environment Difficult to scaleCorrect post-translational modification Few labelling strategies availableMost GPCRs expressed are active

Cell-free expression Only the protein of interest is produced Poor scalabilityCan express toxic proteins Yields >1 mg/ml reactionCan include detergents and membrane mimetics May require optimization of detergents and membrane mimeticsRelatively expensive Low success rate of GPCR expression to dateComplete labelling control

into a polypeptide, protein or ligand of limited positions. Itrequires solid-phase peptide synthesis or chemical synthesis. Thisapproach has been used extensively to study amyloid peptides,membrane peptides and GPCR ligand conformation [46,64,147–151]. Selective labelling, including forward and reverse labelling,refers to the biosynthetic incorporation of a single type or severaltypes of labelled amino acid(s) with the rest unlabelled into theprotein expression media. Selective labelling is widely used forthe protein resonance assignments and structure calculations inSSNMR. For example, a selective and extensive labelling schemeusing [1,3-13C]- and [2-13C]-glycerol as the sole carbon sourcein the bacterial growth medium has been used to determine theα-spectrin SH3 (Src homology 3) domain, β-crystallin and outermembrane protein G [152,153].

A selective labelling scheme has also been used in the HEK-293 system to express labelled rhodopsin for structure andfunctional studies [49–52,55,141]. The reverse labelling approachhas been designed to study structure and dynamics of the sensoryrhodopsin II and the K+ ion channel KcsA–Kv1.3 [74,154]. Thisapproach has the advantage of decreasing the large numbersof overlapping resonances from the hydrophobic TM α-helices,making the extensive assignments a reality. By taking advantageof the glycolysis pathway, the TEASE (ten amino acid selectiveand extensive labelling) 13C selective labelling scheme has been

suggested to probe the TM segments of membrane proteins [155],and this reverse labelling approach may have the possibility ofbeing adapted for other different applications for its flexibility.Recently, a novel selective labelling scheme has been proposed byusing [2,3-13C,15N]-labelled phenylalanine and tyrosine residuesand fully labelled glycine and alanine residues to restore thefavourable cross-relaxation properties of the glycerol samplesin order to obtain inter-residue cross-peaks [91]. This labellingscheme has several advantages: (i) it is composed of a low numberof small isolated spin systems; (ii) transfer of magnetization intothe side chain is thus eliminated and spectral quality enhanced;and (iii) the number of inter-residue cross-peaks is significantlyincreased, which is important for both assignment and structurecalculation [91]. This labelling scheme or a similar one may beapplicable to other large membrane proteins, such as the 7TMfamily proteins.

Uniformly labelling schemes are the simplest and most cost-effective biosynthetic labelling method for protein SSNMR.Normally, uniformly 13C-labelled glucose or glycerol and 15N-labelled ammonium chloride or ammonium sulfate are used asthe labelled precursors in the growth medium. With a singlesample, all of the structural constraints can be obtained througha set of correlation experiments, and the protein structure can becalculated thereby. This approach has been first demonstrated on



Figure 2 Schematic representation of different sample environments for NMR studies of GPCRs

Reprinted by permission from Macmillan Publishers Ltd: Biochimica et Biophysica Acta [Satita Tapaneeyakorn, Alan D. Goddard, Joanne Oates, Christine L. Willis, Anthony Watts. Solution- andsolid-state NMR studies of GPCRs and their ligands (2011) 1808(6), 1462–1475], c© 2011.

the microcrystalline proteins of known structure [156–159], andthen successfully applied to membrane protein for the structuredetermination [40,74,81,85,160–162].

By taking the advantage of minimizing the 1H–1H dipolarcouplings caused spectral broadening, predeuteration of proteinshas been exploited to gain even higher resolution for U-13C-and U-15N-labelled proteins in SSNMR, and this approachhas also been attempted to express the fully labelled 7TMprotein bacteriorhodopsin for the structure investigation [163].However, with an increase in deuteration level, protein expressionlevel may fall and it may even be difficult to grow somestrains on 2H2O [97]. Therefore a good balance needs to beconsidered.

Choosing an appropriate sample environment is critical notonly for generating a high-resolution NMR spectrum, but alsofor the protein to have correct folding and activity. Obtaining ahomogeneous sample preparation leads to improved linewidthsand therefore spectral resolution, whereas heterogeneous samplescan result in artefacts such as unexpected peaks and peak doubling[97]. Furthermore, it is not enough to show that a protein constructis functional to validate a structure unless the functional assay isperformed in the same environment as that used for the structuralcharacterization [164]. For small proteins, nano/micro-crystallineor nanodisc samples have been proved to be a good choice foryielding high-quality spectra in solids [165–168]. For membraneproteins, samples can be prepared in detergent micelles, bicellesor lipid bilayers. Given that membrane proteins function withina bilayer environment, it is more biologically applicable to beable to carry out structural investigations in lipids [97]. Structuraldata obtained in an appropriate lipid bilayer environment canserve as benchmarks for validating structures determined inother mimetic environments [164]. Figure 2 illustrates somecommonly used sample environmental forms for NMR studies ofGPCRs [97].

LIGAND CONFORMATION AND BINDING

The molecular mechanisms of GPCR activation are of centralinterest in cellular responses to biogenic stimulus and drugs.However, the direct atomic resolution information on theinteractions of GPCR with its ligand is available throughX-ray crystallography only for a limited number of receptors,whereas, for the great majority of them, indirect methods ofanalysis have been used. Knowledge of the three-dimensionalstructures of several GPCRs, such as bovine rhodopsin [8–10], β1AR and β2AR [11,12], A2a adenosine receptor [13],CXCR4 chemokine receptor [14], dopamine D3 receptor [15], thehuman histamine H1 receptor [16], the M2 and M3 muscarinicacetylcholine receptors [17,18], the human κ-opioid receptor[19], the nociceptin/orphanin FQ receptor [20], the μ-opioidreceptor [21] and the neurotensin receptor [22], have beenresolved by X-ray crystallography in either an inactive stateor and agonist/antagonist-bound form at high resolution, whichopens up new possibilities for investigating GPCRs of humantherapeutic significance and for rational drug design. However,despite the availability of those crystal structures, achieving adeep understanding of GPCR activation has still been challengingbecause of the lack of high-resolution structure at atomiclevel for the activated state and the details of protein–ligandinteractions. For example, structures of bovine rhodopsin in aphotointermediate state and Meta II active state, the ligand-free opsin and bound form with Gα-CT peptide have beensolved within the last few years [38,169–172], but the activationmechanism is still not very clear, especially when it is relatedto the constitutive activity caused by single mutation andretinitis pigmentosa. In this context, SAR (structure–activityrelationship) analyses [173] and drug discovery studies wouldbenefit greatly from some other experimental probes of receptor–ligand interactions, such as those that SSNMR could provide.



Figure 3 Comparison of retinal chemical shifts in rhodopsin different photointermediate states

The chemical structures of retinal in (a) 11-cis and (b) all-trans configuration. (c) Chemical shifts of the retinal atoms in ground state, and Batho-, Meta I and Meta II photointermediate states. Thechemical shift data are collected from [181–183] (black), [58] (blue), [178–180] (green) and [53,184] (red).

SSNMR spectroscopy has been applied successfully to GPCRs todetect ligand binding and to analyse protein–ligand interactions,using selective isotope-labelled specific residues and the ligand.

The conformation changes of retinal chromophore andrhodopsin activation upon the 11-cis to all-trans isomerization ofretinal have been studied extensively by MAS SSNMR throughchemical shift measurements, distance measurements, correlationexperiments and 2H-NMR [46,49–54,56–60,62,63,174–180].Complete assignment of the retinal carbons in ground state andpartially in the Batho-, Meta I and Meta II intermediates havebeen achieved [53,58,178–184]. These valuable data allow usan insight into how the conformational changes of the retinal–PSB (protonated Schiff base) complex are upon isomerizationand binding, and how the changes lead to the activation ofrhodopsin through the transition from the ground state to Meta IIintermediate eventually (as shown in Figure 3).

Large downfield and upfield changes in chemical shifts havebeen observed at the C-16 and C-17 sites of the β-iononering, and on the retinal polyene chain around the C-9–C-10–C-11–C-12–C-13–C-14–C-15 region (Figure 3). A largeupfield shift of C-13 from the ground state to the Meta IIstate may be attributed to the break of the Glu113 salt bridgeas the counterion due to the displacement of the helix uponactivation. Swapped chemical shift positions of C-16 and C-

17 may be attributed to the local hydrophobic environmentalchange due to the interaction of the helices with the β-ionone ring at the binding pocket upon retinal isomerization.Upfield shifts of C-9 and C-11 and downfield shifts of C-10,C-12 and C-14 of the retinal polyene chain may be attributed tothe changes of the polyene chain configuration due to the 11-cisto all-trans isomerization and some possible interactions at thebinding pocket upon the displacements of the helices.

Two-dimensional PDSD (proton-driven spin diffusion)experiments have been used to study the human histamine boundto the H1 receptor with the 13C chemical shifts assigned by DQF(double quantum filtration) experiments to probe changes in theprotonation state of the ligand histamine binding to the receptor[66], as shown in Figure 4. Two ligand protonation states havebeen identified and provided the basis of an activation mechanisminvolving proton transfer from the ligand to the receptor.

In addition, the structure of neurotensin bound or unbound to theNTS1 receptor has also been investigated by SSNMR, revealinga β-strand conformation upon binding [64]. The conformationof the bradykinin peptide bound to the human bradykininB2 receptor in DDM (dodecyl maltoside), on the other hand,has been proposed to hold a double-S-shape structure [185].These type of studies can be also conducted to rationalize thebiological activities of compounds on the basis of their bioactive



Figure 4 Olefinic and aromatic regions of a 13C PDSD two-dimensional spectrum of histamine bound to the H1 receptor at ∼90 % ligand saturation

Assignments of the monocationic and dicationic histamine species are shown in blue and red respectively. The top traces in grey in the upper panels represent the resonances in a DQF one-dimensionalspectrum collected from the same preparation. The DQF spectrum only presents signals from 13C nuclei that have another 13C as a neighbour. In this way the natural abundance background responseis suppressed. The primed numbers represent the contribution from the dicationic species. Reprinted with permission from Ratnala, V.R.P., Kiihne, S.R., Buda, F., Leurs, R., de Groot, H.J.M. andDeGrip, W.J. (2007) Solid-state NMR evidence for a protonation switch in the binding pocket of the H1 receptor upon binding of the agonist histamine. J. Am. Chem. Soc. 129, 867–872. c© 2007American Chemical Society.

conformations, as has been done in the case of two natural peptidesactive at the neuropeptide NPR-1 receptor [186].

STRUCTURAL ACTIVATION UPON LIGAND BINDING

Upon binding of agonists, which typically occurs in closeproximity to the extracellular opening of the helical bundle,GPCRs undergo a series of structural changes that cascade fromthe extracellular to the intracellular part of the receptor andultimately lead to G-protein activation. The global toggle switchcoupled with several highly conserved residues switching betweensubstantially different conformations in the active comparedwith the inactive state, as molecular microswitches, triggersthe conformational changes for activation and formation of theG-protein-binding site of the receptors [27,30,33]. In the last fewyears, several works has been focused on the SSNMR studies ofthe structural changes on the extracellular side of the receptorcaused by retinal isomerization. By combined SSNMR chemicalshift measurements and two-dimensional DARR (dipolar-assistedrotational resonance) [187] NMR measurements with a selectivelabelling scheme and mutagenesis, coupling of the retinalisomerization with the displacement of EL2 on rhodopsinactivation has been investigated extensively [52–55]. Figure 5

shows the two-dimensional 13C DARR NMR spectra of retinal–EL2 interactions. Close contacts have been observed betweenthe retinal 13C-14 and 13C-15 and 13Cβ-Ser186 (Figure 5a), theretinal 13C-12 and 13C-20 and 13C-1-Cys187 (Figure 5b), andthe retinal 13C-12 and 13C-20 and 13Cα-Gly188 in rhodopsin(Figure 5c). But the contacts between the retinal 13C-9 and 13C-12and [U-13C6]Ile189 in rhodopsin or Meta II were not observable(Figure 5d) [54]. All of the results indicate that the formation ofMeta II is accompanied by the displacement of EL2 away from theretinal-binding site and there is a rearrangement in the hydrogen-bonding networks connecting EL2 with the extracellular endsof TM α-helices IV, V and VI. This displacement is coupledto the rotation of TM α-helix V and breaking of the ioniclock connecting TM α-helix III and TM α-helix VI [54]. Thesecomprehensive results may lead to further investigation of themolecular mechanism of the cavity formation between TM α-helices III, V and VI for the G-protein binding [188].

THREE-DIMENSIONAL STRUCTURE DETERMINATION

Determination of GPCR structures is still a frontier of structuralbiology. Over the last few years, many progresses have beenmade from NMR methodology to sample preparation in order



Figure 5 Two-dimensional 13C DARR NMR spectra of retinal–EL2 interactions

Rows from the two-dimensional 13C DARR NMR spectra of rhodopsin (black) and Meta II (red) are shown. The rhodopsin crystal structure (grey) with the Meta II model (orange) obtained frommolecular dynamics simulations is shown in the centre. Reprinted by permission from Macmillan Publishers Ltd: Nature Structural and Molecular Biology (Ahuja, S., Hornak, V., Yan, E.C.Y., Syrett,N., Goncalves, J.A., Hirshfeld, A., Ziliox, M., Sakmar, T.P., Sheves, M., Reeves, P.J., Smith, S.O. and Eilers, M. (2009) Helix movement is coupled to displacement of the second extracellular loop inrhodopsin activation, 16(2), 168–175, c© 2009.

to reduce the dipolar truncation, establish reliable short- andlong-range distance constraints, and improve spectral linewidthsand resolution for a full structure determination. However, signaloverlapping and fast longitudinal and transverse relaxation, whichcause the line broadening and signal intensity loss in manycorrelation experiments, are still the main problems that impedethe pace of SSNMR structure determination of GPCRs.

PISEMA (Polarization Inversion Spin Exchange at the MagicAngle) [189] is a powerful SSNMR method for the determinationof membrane protein structure and conformation through high-resolution separated local field spectra, where each resonancefrom a 15N-labelled amide site in the protein backbone ischaracterized by orientation-dependent heteronuclear 1H–15Ndipolar coupling and 15N chemical shift frequencies. Helices resultin characteristic PISA (Polarity Index Slant Angle) [190,191]wheel patterns of resonances that reflect their tilt and polarityin the bilayers. From such wheels, it is possible to determinethe atomic-resolution secondary structure, the tilt angles ofα-helices or β-sheets, and, in favourable cases, sequentialassignment of all resonances on the basis of a single uniqueresonance assignment [192–194]. Figure 6(a) shows a schematicrepresentation of CXCR1 structure, and Figure 6(b) shows a two-dimensional PISEMA spectrum of selectively [15N]Ile-labelledCXCR1 in magnetically aligned bicelles oriented perpendicularto the direction of the magnetic field. The spectrum demonstratesthe complementary use of selective isotopic labelling, whichis particularly valuable in this context because the mapping ofsecondary structure on to the experimental spectra assists theassignment and structure determination processes [195]. Theability to obtain spectra with single-site resolution as shown inFigure 6 implies that GPCRs in phospholipid bilayers are suitablesamples for structure determination by SSNMR. Moreover, it ispossible to utilize these spectra for screening of small-moleculelibraries and the application of SARs by SSNMR [173] for thedevelopment of drugs that target GPCRs. The three-dimensionalstructure of human CXCR1 has just been reported in Nature withimportant features for intracellular G-protein activation and signal

Figure 6 Solid-state NMR spectra with single-site resolution of CXCR1

(a) CXCR1 model in bilayers. (b) Two-dimensional PISEMA spectrum of selectively[15N]Ile-labelled CXCR1 in magnetically aligned bicelles oriented perpendicular to the directionof the magnetic field. The blue circled contours arise from residues in TM α-helices, whereasred circled resonances are from residues in loop and terminal regions which are likely tohave irregular structures. These results demonstrate that GPCRs in phospholipid bilayers aresuitable samples for structure determination by SSNMR. The spectra also enable studies ofdrug–receptor interactions. Reprinted with permission from Park, S.H., Prytulla, S., De Angelis,A.A., Brown, J.M., Kiefer, H. and Opella, S.J. (2006) High-resolution NMR spectroscopy of aGPCR in aligned bicelles. J. Am. Chem. Soc. 128, 7402–7403. c© 2006 American ChemicalSociety.

transduction in liquid crystalline phospholipid bilayers withoutmodification of its amino acid sequence and under physiologicalconditions [196].

GPCR DYNAMICS

Transmission of signals between cells, within cells and fromthe extracellular environment to the cellular interior is essentialto life, and the dynamic properties of the signalling proteinsare crucial to their functions [197]. Although the influence ofstructure in molecular recognition and catalysis by proteins iswell appreciated, the role of dynamics is largely unknown andoften ignored [198]. It has been proved that the internal proteindynamics can potentially affect protein function through a variety



Figure 7 B factors along the rhodopsin sequences for both dimers

B factors are taken from [8]. The positions of the TM α-helices (shading) and CP loops are indicated. �, Crystal 1, �, crystal 2. Reprinted with permission from Judith Klein-Seetharaman, Dynamicsin rhodopsin, ChemBioChem 3, 981–986, Wiley c© 2002 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figure 8 Solid-state 2H-NMR captures site-specific changes in picosecond–nanosecond dynamics of retinal during light activation of rhodopsin

Comparison of the spin-lattice (T 1Z ) relaxation times of the retinal methyl groups in the same state in rhodopsin (a–c) and the same methyl group in the different photointermediate states (d–f).Arrhenius-type plots of the T 1Z data against inverse temperature for the retinylidene ligand in the dark, Meta I and Meta II states show that the T 1Z times for the C-9 and C-13 methyl groups increasewith temperature, whereas those for the C-5 methyl group show a distinct minimum. The light-induced changes in the retinylidene ligand dynamics are encapsulated by pronounced T 1Z differences inthe Meta I and Meta II states. (a–c) Reproduced with kind permission from Struts, A.V., Salgadoc, G.F.J., Brown, M.F. (2011) Solid-state 2H NMR relaxation illuminates functional dynamics of retinalcofactor in membrane activation of rhodopsin. Proc. Natl. Acad. Sci. U.S.A. 108(20), 8263–8268. c© 2011. (d–f) Reprinted by permission from Macmillan Publishers Ltd: Nature Structural andMolecular Biology [Andrey V. Struts, Gilmar F.J. Salgado, Karina Martınez-Mayorga, Michael F. Brown (2011) Retinal dynamics underlie its switch from inverse agonist to agonist during rhodopsinactivation, 18(3), 392–394], c© 2011.



Figure 9 Illustration of the local and global dynamics of the chemokine receptor CXCR1 characterized by SSNMR powder patterns of both carbonyl carbonand amide nitrogen chemical shift of unoriented samples containing 13C- and 15N-labelled CXCR1

Reprinted with permission from Park, S.H., Casagrande, F., Das, B.B., Albrecht, L., Chu, M. and Opella, S.J. (2011) Local and global dynamics of the G protein-coupled receptor CXCR1. Biochemistry50, 2371–2380. c© 2011 American Chemical Society.

of mechanisms, some of which are obvious in nature, whereasothers are subtle and remain to be fully explored and appreciated.Therefore understanding the dynamic nature of a GPCR is crucialto reveal its function and activation.

The B factors of the atoms in the rhodopsin crystal dimer fromX-ray diffraction [199] are shown in Figure 7. There is largevariation in B factors at the different positions along the sequence,with greatly increased disorder in loop regions. This observationsuggests the presence of a gradient in mobility along the TMdomain from the NT ends of the helices towards the CT ends,with maximum mobility in the CLs, which may indicate that theconformational fluctuations in dark-adapted rhodopsin probablyhaving a functional significance and probably allowing light-induced conformational changes that are part of the signallingprocess to occur [199].

The molecular mechanism of rhodopsin activation remainsunknown as atomic resolution structural information for the activeMeta II state is currently lacking [32]. The light-activated Meta IIstate is transient and cannot form high-resolution diffraction data[38]. The crystal structure of opsin in ligand-free form [170] andbound with Gα-CT peptide [171] does not contain the activatedligand and its activity does not match rhodopsin.

SSNMR may be the best technique to study membrane proteindynamics in its native lipid environment through the anisotropicspin interactions and spin relaxation; many methods, coupled withnovel labelling schemes, have been explored to probe membraneprotein dynamics over a wide range of timescales [83,93,200].For example, site-specific 2H labelling is the most commonlyused scheme for labelling methyl groups of alanine, leucine andvaline, and is an excellent probe of the protein local dynamicfluctuation.

The retinal methyl groups play an important role in rhodopsinfunction by directing conformational changes upon transition intothe active state [201]. Site-specific 2H labels have been introducedinto the C-5, C-9 or C-13 methyl groups of retinal and 2H-SSNMRmethods applied to obtain order parameters and relaxation timesto elucidate picosecond-to-nanosecond-timescale motions of theretinal ligand that influence larger-scale functional dynamics of

rhodopsin in the inactive dark state, as well as photo-intermediateMeta I and Meta II states in membranes [62,63], as shownin Figure 8. Analysis of the angular-dependent 2H-NMR lineshapes for selectively deuterated methyl groups of rhodopsin inaligned membranes enables determination of the average ligandconformation within the binding pocket. The relaxation datasuggest that the β-ionone ring is not expelled from its hydrophobicpocket in the transition from Meta I to active Meta II state.The Meta I to Meta II transition involves mainly conformationalchanges of the protein within the membrane lipid bilayer ratherthan the ligand. The dynamics of the retinal methyl groups uponisomerization are explained by an activation mechanism involvingco-operative rearrangements of EL2 together with TM α-helicesV and VI. These activating movements are triggered by stericclashes of the isomerized all-trans-retinal with the β4 strandof the EL2 and the side chains of Glu122 and Trp265 within thebinding pocket. A multiscale activation model has been proposedby 2H-SSNMR spectroscopy, suggesting that retinal initiatescollective helix fluctuations in Meta I and Meta II equilibriumon the microsecond-to-millisecond timescale which is differentfrom the standard model of a single light-activated receptor (R∗)conformation that yields the visual response.

The local and global dynamics of the chemokine receptorCXCR1 has been characterized by using a combination of solutionand SSNMR experiments, revealing that the essential dynamicsof CXCR1 consist of fast local backbone motions for residuesnear the NT and CT, but not for residues in the TM α-helicesor interhelical loops, and rapid global rotational diffusion of theprotein about the bilayer normal in liquid crystalline, but not gel-phase, phospholipid bilayers [202], as illustrated in Figure 9.

CONCLUSIONS AND FURTHER PERSPECTIVES

MAS SSNMR has made tremendous progress showing itscapability of determining membrane protein structure, ligandbinding and protein dynamic conformation on a varietyof timescales at atomic resolution. Despite the numerous



technical challenges, SSNMR spectroscopy has the potentialto become a leading technique in the study of the structure–function relationships of GPCRs and their interactions withligands. In contrast with crystallographic methods, whichprovide high-resolution, but static, molecular snapshots, NMRtechniques can provide dynamic pictures of receptor structures,receptor–ligand interactions, and reaction intermediates and theirenergy landscapes, thus offering insights into the molecularmechanisms of ligand recognition, receptor activation andsignalling mechanisms.

Recently, by transferring the polarization of electron spinsto nuclei, the MAS DNP (dynamic nuclear polarization) hasbeen successfully applied to study of the intermediate statesof a 7TM light-driven proton pump, bacteriorhodopsin. Theenhanced sensitivity of DNP permitted the characterization ofthe retinal conformation in the K, L and M states [78,203,204]for the first time in SSNMR. The introduction of DNP,as well as other advances in SSNMR technology, such asshortening the spin longitudinal relaxation time T1 by Cu-EDTA doping [205] and employing the PACC (paramagneticrelaxation-assisted condensed data collection) technique [206],together with progress in the preparation of GPCR samples,are expected to lead in the future to the determination ofthe three-dimensional structure of whole GPCRs, to identify thedynamic switch of GPCRs between an inactive (R) state andan active (R∗) conformation [207], and to provide furthermechanistic insights into the complex cascade of motions andrearrangements characteristic of the activation process. Further-more, these methodological advances are also likely to foster theapplication of SAR by NMR techniques [173] to the discoveryof lead compounds for GPCRs through NMR-based high-throughput screening and their subsequent structure-basedoptimization [208–210].

FUNDING

Financial support to A.W. from the Medical Research Council and Engineering and PhysicalSciences Research Council, and the State Administration of Foreign Experts Affairs, P.R.China, through the High-end Foreign Experts Recruitment Program [grant number GDW20123100086] are very much appreciated.

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Received 26 October 2012/13 December 2012; accepted 18 December 2012Published on the Internet 28 February 2013, doi:10.1042/BJ20121644


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