Illuminating the energy landscape of GPCRs: the key contribution of

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Illuminating the energy landscape of GPCRs: the key contribution of solution-state NMR associated with Escherichia coli as an expression host Marina Casiraghi, Marjorie Damian, Ewen Lescop, Jean-Louis Baneres, and Laurent J Catoire Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.8b00035 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 2, 2018

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Biochemistry

Illuminating the energy landscape of GPCRs: the key contribution of solution-state NMR associated with Escherichia coli as an expression host Marina Casiraghi,† Marjorie Damian,‡ Ewen Lescop,§ Jean-Louis Banères,*,‡ Laurent J. Catoire,*,† 5

†

Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, UMR 7099, CNRS/Université Paris Diderot, Sorbonne Paris Cité, Institut de Biologie Physico-Chimique (FRC 550), 13 rue Pierre et Marie Curie, 75005 Paris, France; ‡ Institut des Biomolécules Max Mousseron (IBMM), UMR5247 CNRS – Université Montpellier – ENSCM, UFR pharma§ cie, 15 av. Charles Flahault, 34093 Montpellier, France; Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Sud, Université Paris-Saclay, 1, av. de la Terrasse, 91198 Gif-sur-Yvette, France

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ABSTRACT: Conformational dynamics of GPCRs are central to their function but are difficult to explore at the atomic scale. Solution-state NMR has provided the major contribution in that area during the past decade, despite non-optimized labeling schemes due to the use of insect cells, and to a lesser extent yeast, as the main expression hosts. Indeed, the most efficient isotope-labeling scheme ever to address energy landscape issues for large proteins or protein complexes relies on the use of 13CH3 probes immersed in a perdeuterated dipolar environment, which is essentially out of reach of eukaryotic expression systems. In contrast, although its contribution has been underestimated because of technical issues, Escherichia coli is by far the best-adapted host for such labeling. As it is now tightly controlled, we show in this review that bacterial expression can provide a NMR spectral resolution never achieved in the GPCR field.

INTRODUCTION G protein-coupled receptors (GPCRs) are major players in virtually all aspects of physiological processes. The large number of biological functions they control also makes them one of the most prominent families of pharmacological targets in biomedicine.1 The current model of signal transduction states that the dynamics of GPCRs govern their signaling, as the absence of a unique rigid conformation confers a plasticity that allows the receptor to adopt multiple states linked to distinct functional outcomes.2 Mapping out the conformational landscape of GPCRs is therefore the subject of intense research, as it is required to get a clear picture of how GPCRmediated signaling occurs. High-resolution crystal structures of several GPCRs have been solved during the last decade, giving snapshots at the beginning and end of the activation process of these receptors.3 This represents a precious starting point to further explore the energy landscape of these complex allosteric machines.4,5 However, additional biophysical studies are still required to complete the description of the conformational space these receptors can span and the chemical exchanges between these different sub-states. Among those, solution-state NMR represents a powerful technique to address important issues in the energy landscape of biomolecules.6 However, to get the most out of this technique with large tumbling objects like GPCRs in membrane-like environments, it needs to be associated with specific isotope-labeling schemes, in addition to high magnetic fields and appropriate methodologies. Such schemes rely on introducing perdeuterated protonated and 12C2H and 13C-labeled methyl probes within a perdeuterated receptor.7 So far, Escherichia coli (E. coli) represents the best host available to produce perdeuterated (i.e. >98%) recombinant proteins. Different approaches have therefore been developed for expressing GPCRs in bacterial systems that include addressing the receptor into the membrane,8 directed evolution to increase the receptor stability,9 or accumulation in inclusion bodies followed by in vitro folding and subsequent reconstitution of the receptor in lipid nanodiscs.10 The latter strategy, summarized in Figure 1, appears particularly attractive as it allows the most relevant isotope-labeling schemes, a strict control of the lipid composition and provides unmodified fully functional receptors, based on their ability to bind ligands and activate their cognate signaling proteins. We discuss here the impact of this strategy for the development of state-of-the-art NMR studies.

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Figure 1. Strategy to obtain high resolution NMR data of fully active and stable GPCRs in a lipid environment.

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DESIGN, PRODUCTION AND PURIFICATION OF GPCRs EXPRESSED IN ESCHERICHIA COLI INCLUSION BODIES Overexpression of membrane proteins (MPs) in a functional form in the plasma membrane of a given host is more like a via dolorosa than an easy task because of the limited volume afforded by the membrane11 of the host and to toxic effects associated with saturation of the MP insertion machinery. Different strategies have been developed to circumvent these drawbacks. One of them relies in a directed evolution method that allows direct selection of more stable, although modified, GPCRs and has been applied to the neurotensin receptor NTS1.9 Other alternatives, like cell-free in vitro synthesis or targeting of expressed MPs to inclusion bodies (IBs) have also been developed. Successful examples of production of functional GPCRs in cell-free systems are limited.12-14 Regarding accumulation in IBs, these have the advantage to be easy to purify and to yield tens of mg of proteins per liter of culture. This is particularly relevant in situations where labeling strategies have an impact on the yield of expression, as in the case of perdeuteration that requires growth in 100%-D2O solutions in the presence of deuterated glucose and, eventually, of additional deuterated amino acid precursors. GPCR accumulation in IBs usually requires the receptor to be fused to an additional protein such as Glutathione Stransferase (GST),15 KetoSteroid Isomerase (KSI)16 or a fragment of the α subunit of integrin α5β1.17 Importantly, the latter allows high levels of expression, independently of the receptor considered, which is not the case of other fusion partners.17 To be noted, receptors produced in E. coli are devoid of all post-synthetic modifications GPCRs can undergo (phosphorylation, glycosylation, palmitoylation). However, most of these modifications are involved in membrane export and addressing to specific compartments,18,19 and/or in the recruitment of signaling partners.20 Hence, they likely do not dramatically impact on the conformational dynamics of the TM domains that are the main focus of the NMR-based analyses. For instance, phosphorylation has been shown to essentially affect the dynamics of the C-terminal region of the β2-adrenergic receptor (β2AR).21 Accordingly, in most NMR studies with GPCRs expressed in hosts that can address post-synthetic modifications (sf9, P. pastoris), the C-terminal tails of the receptors where the phosphorylation and palmitoylation sites are located were trimmed while the glycosylation sites were mutated. Besides, it must be also kept in mind that different strategies have been developed to introduce phosphorylated22,23 or palmitoylated19 mimics into proteins expressed in E. coli that could be used to overcome this limitation. In addition, as far as phosphorylation is considered, the purified receptors in nanodiscs can also be phosphorylated in vitro with recombinant GRKs even when the corresponding region is expressed in E. coli, as recently shown with the β2AR.21

IN VITRO FOLDING OF GPCRs Targeting the expression of receptors to IBs greatly improves the protein yields (see ref. 10 for a comparison of the expression yields in inner membranes and IBs). However, this requires to subsequently fold them to their native states. Since the pioneering work on folding of bacteriorhodopsin,24 more than 50 α-helical and almost 40 β-barrel, monomeric and oligomeric MPs, either prokaryotic or eukaryotic, have been folded or refolded in vitro during the last three decades (see ref. 25 for an inventory). The starting unfolded state appears to be also important for a successful folding. As denaturing conditions, sodium dodecyl sulfate (SDS) detergent appears as a very helpful and convenient medium. Indeed (i) it does not lead to a complete random-coil state but rather favors formation of α-helices26 with a significant degree of overlap between SDS-denatured conditions and native transmembrane α-helices, and (ii) it can be removed almost instantly by adding KCl which leads to its insoluble potassium salt KDS. A rapid transfer from denaturing to non-denaturing conditions is presumably better to avoid conformations that may correspond to a local free-energy minimum that would not reflect the native state, or conformational ensemble, of the protein.25 Accordingly, folding of GPCRs from a SDS solution to


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detergent solutions,27 bicelles,16 amphipols28 or nanodiscs29 has been repeatedly described. For instance, following a procedure that was based on folding in the presence of amphipols and lipids (DMPC) and a subsequent transfer to nanodiscs, the low affinity leukotriene receptor BLT2 was obtained in an about 95% active state with regard to ligand binding.30 The folded receptor was also able to catalyze the activation of its cognate G protein Gαi with EC50 values in the very same range than those reported in the literature for the same receptor in a cellular environment.31 Moreover, GTPγS binding rates were very similar to those reported for other GPCRs purified from eukaryotic expression systems under a native form32 or even from natural tissues.33 In this context, caution needs to be exerted when considering the amount of ligand-competent receptor only, as ligand binding may force the observed conformation towards the native one. Hence, the EC50 values have also to be considered and compared to the values inferred for the same receptor in living cells, as this is likely a more faithful diagnostic of a native conformational ensemble. Accordingly, NMR investigations of BLT2 conformational ensemble in various situations confirmed that BLT2 was not kinetically blocked in a conformation that would not be a free-energy minimum.30 The same situation was encountered with the ghrelin receptor GHSR that adopted a fully functional state after transfer from SDS-solutions to POPC/POPG nanodiscs through an amphipol-mediated folding step.34 In this case also, the purified receptor binds ligands, activates G proteins and recruits arrestin-3 in a ligand-dependent manner totally related to what is observed in mammalian heterologous systems.34 Again, all these evidences point towards a folded state, or conformational ensemble, of the purified receptor very similar to that it adopts in a native membrane. In contrast to BLT2, however, getting a homogeneous fraction of fully functional GHSR required an additional affinity chromatography purification step on an immobilized ligand, as refolding yielded about 40-50% of fully functional receptor.34 The availability of purification methods based on ligand-affinity chromatography opens the way to the use of bacterially expressed receptors in cases where refolding is not quantitative. It is to be emphasized, however, that such a purification step is not restricted to procedures involving folding from IBs, as solubilization from membrane fractions with detergents is, in many cases, associated with some receptor unfolding. Accordingly, a ligand-affinity purification step is also required to get homogeneous fractions for several GPCRs recovered from membranes of diverse expression hosts.35,36 Overall, our data largely indicate that it is totally possible to fold GPCRs back to their native state when starting from IBs, even if the recovery yields depend on the receptor considered. So far, we have no direct mechanistic model to account for this refolding efficiency. Indeed, as stated above, very different yields have been obtained for the different receptors we tested (e.g. BLT2 vs. GHSR). Whether the low yield obtained for the ghrelin receptor is the consequence of its higher conformational dynamics,37 or from a more general point of view, whether the efficiency of refolding into lipid nanodiscs is dependent of the receptor thermodynamic stability is an open question at the present stage.

SELECTIVE LABELING OF 13CH3 AT RESIDUES-SPECIFIC METHYL SITES IN A PERDEUTERATED RECEPTOR FOR NMR STUDIES

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Studying large proteins or protein complexes by NMR with only the naturally abundant 1H nucleus is not realistic because of spectral crowding due to the limited dispersion of 1H chemical shifts, in addition to unfavorable relaxation properties that render 1H signals broad because of the predominant influence of the overall tumbling rate. To overcome these drawbacks, multi-dimensional NMR, based on the isotope enrichments in 15N and 13C nuclei and perdeuteration has been introduced. 15N and 13C nuclei, which have the property to have chemical shift spectral widths larger than 1H, improve the distinction of signals by developing the observation in additional spectral dimensions. Perdeuteration is used in addition to slow down the relaxation rates of the nuclei under investigation by weakening the dipolar interactions with neighboring protons. This results in a spectacular gain in resolution for large proteins.7 When associated with high magnetic field and appropriate pulse sequence methodology, working with protonated and 13C-labeled methyl groups immersed in a perdeuterated environment actually represents the most powerful labeling scheme for studying large proteins.38-41 Indeed, in addition to the presence of three protons that contributes to sensitivity, the rotation of the methyl head renders protons chemically equivalent and generally CH3 groups are located at extremities of amino acid side-chains, two aspects which increase the signal detection compared for instance to amide protons located along the backbone of the protein, these latters, however, when associated with 15N nuclei, can still represent a viable alternative in some cases. In addition, especially in the case of membrane proteins, methyl groups are located along the trans-membrane part which makes them interesting probes to sample conformational dynamics when a signal is transmitted from one side of the membrane to the other. Selective labeling of 13CH3 at residue-specific methyl sites in a perdeuterated (i.e. > 98%) protein is best achieved using E. coli as expression host.42 Indeed, amino acid metabolic pathways are well known for this microorganism. These pathways can be carefully tuned to produce 13CH3-labeling at residue specific methyl sites, including the possibility to stereo-specifically label prochiral methyl groups of Leu and Val, for instance.43 This means also that, when correctly controlled, no isotope scrambling or dilution occurs and high yields of selectively labeled proteins are produced at a reasonable cost. Hence, E. coli represents a unique organism for its genetic engineering and for its capacity to grow in very hostile conditions, like 100%-D2O solutions. Other organisms can be used to produce deuterated heterologous proteins, like insect,44 yeast45 or mammalian cells46 for instance. However, they do not allow the production of perdeuterated proteins, which is a major drawback in the case of large systems, as even a residual of few percent of neighboring protons around nuclei under investigations by NMR impacts dramatically on their relaxation properties, and so, on the quality of the NMR signal7,42,47 (vide infra Figures 2 and 3). In order to improve the dipolar environment of selectively methioninelabeled GPCRs using insect cell/baculovirus expression systems, the group of Shimada developed an original strategy which consists to add commercially available deuterated amino acids and algal amino acid mixtures in addition to 13CH3-ε-methionines to an amino acid deficient medium. Following this protocol, fourteen types of amino acid residue could be deuterated at level comprised between 50 and 90% with the β2AR.44 To be noticed, this method requires an experimental 3D atomic structure or a model of the receptor under consideration in order to select the amino acids around the methionines inside the receptor to minimize the number of deuter-


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ated amino acids injected into the medium because the latter substantially affect protein production yields. Despite all these efforts, the remaining protons around the methyl group of Met residues still leads to broader signals compared to the bacterial approach discussed herein (vide infra Figure 2C). Some yeast expression systems have also been reported to be useful for high-level of deuteration (~90%) and could represent a valuable alternative to the approach described herein,48,49 even though, regarding the efficiency of incorporation of labeled-precursors (estimated for instance to 45% for the deuterated alpha-ketobutyrate49) and the production of integral membrane proteins, yields are probably much lower at an equivalent cost. Other organisms, like some algae, can grow in 100%-D2O solutions,50 but producing recombinant proteins in these organisms by genetic engineering is either not known or much more complicated than in E. coli. Moreover, in contrary to lipids and carbohydrates for which their synthesis is almost universal, amino acid metabolic pathways vary across species, and this renders selective isotope labeling more difficult and not general.

SOLUTION-STATE NMR STUDIES OF GPCR ENERGY LANDSCAPES WITH PROTONATED OR PARTIALLY AND INHOMOGENEOUS DEUTERATED RECEPTORS: CONSTRAINTS AND LIMITATIONS So far, GPCRs conformational landscape has been thoroughly studied by NMR for a few receptors, including the β2AR,44,51-56 the µopioid receptor (µOR),57,58 the β1-adrenergic receptor (β1AR),59,60 the adenosine A2A receptor (A2AR)61-63 and the low affinity leukotriene B4 receptor BLT230 (Table 1). All these studies converge to indicate that GPCRs have a conformational landscape much more complicated that initially thought. Indeed, most of the studies indicate the occurrence of several coexisting states even in the absence of ligands, with, in some cases, several distinct active states with at least one on-activation pathway or intermediate state. However, except for one study,30 the methodology adopted to investigate the conformational ensemble, and sometimes the dynamics, of GPCRs presents a major drawback: the use of fully protonated51-56,58-60 or partially and inhomogeneously deuterated44,57,62,63 receptors. Indeed, traditionally, GPCRs in structural biology are produced in insect cells, which do not allow high level of deuteration, with an efficiency of deuteration that is amino-acid dependent.44 Improved deuteration levels could be recently obtained for GPCRs (~7063 to 90%62) using yeast by progressively adapting the cells to deuterated media, typically during ~7 to 9 days. Even with such high level of deuteration, NMR signals are still significantly broader compared to the bacterial approach (vide infra Figure 3).

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Biochemistry

Table 1. Different approaches for the study of β2AR, μOR, β1AR, A2AR and BLT2 conformational ensembles by solution-state NMR spectroscopy. Receptor

β2AR

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Modifications

Type of isotope labeling

Truncation after Gly365

( CH3)2-Lys reductive

13

Expression system

Environment

NMR experiment

sf9 insect cells

Detergent

2D H, C

di-methylation

β2AR

52

Thermal stabilizing mutation E122W, C-term truncation at residue 348,

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19

C F3-Cys covalently

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13

(β-DDM)

sf9 insect cells

labeled

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Detergent

1D

F NMR

(β-DDM, +CHS)

+Lorentzian deconvolution

Detergent

2D H, C

∆245-259 (ECL3)

β2AR

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Receptor sequence Gly2-Gly365

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ε- CH3-Met

sf9 insect cells

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13

(β-DDM)

β2AR

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β2AR-∆4 Cys deletion

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C F3-Cys265

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Detergent (MNG-3)

1D

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F NMR

+Lorentzian deconvolution β2AR55

β2AR44

Deletion of the 5

ε-13CH3-Met

extra-membraneMet

(3 Met retained)


ε-13CH13-Met

sf9 insect cells

μOR

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β2AR-∆4 Cys deletion

F158W, ∆6 extra-membrane Met

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C19F3-Cys265

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ε- CH3-Met

2D 1H,13C

(β-DDM)

Sf9 insect cells

(5 Met retained) β2AR56

Detergent

Lipid nanodiscs

2D 1H,13C

(POPC + POPG)

sf9 insect cells

sf9 insect cells

Detergent

1D 19F NMR

(MNG-3)


Detergent

2D H, C

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13

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13

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15

(LMNG)

μOR

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M72T mutation

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( CH3)2-Lys reductive

sf9 insect cells

di-methylation

β1AR

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9 thermostabilizing mutations,

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N-Val

Detergent

2D H, C

(MNG-3, +CHS)

sf9 insect cells

3 point mutations, N-term & ICL3

Detergent

2D H, N

(DM)

truncations

A2AR61

β1AR60

V229C mutation


13

C19F3 -Cys229

ε-13CH3-Met

Yeast, Pichia pastoris Detergent

Sf9 insect cells

2 point mutations

A2AR62

N154Q mutation

30

BLT2

N154Q mutation

(MNG-3)


Detergent

2D 1H,13C

(LMNG) δ1-13CH3-Ile

Yeast, Pichia pastoris Detergent 100% D2O

A2AR63

1D 19F NMR

uniformly 15N-labeled

13

Native sequence or mutant with

ε- CH3-Met and

3 extra-membrane Met mutations

δ1- CH3-Ile

13

2

( H-β-DDM)


(LMNG, +CHS)

E. coli in

Lipid nanodiscs

100% D2O

(DMPC + CHS)


2D 1H,13C

2D 1H,15N

1

13

2D H, C

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As a consequence, NMR signals are very broad or undetectable. The situation worsens in the presence of chemical exchanges in timescales that are in the intermediate regime, usually in the ms timescale regarding the magnetic fields used nowadays. Indeed, most of the NMR studies of GPCRs in solution listed in Table 1 indicate the presence of intermediate to slow chemical exchanges at the scale of the 1H, 13C, 15N, 19F NMR chemical shifts. This is why nice resolved NMR signals could be observed with rhodopsin known to be quite rigid64 or receptors that contain several thermostabilizing mutations like in the recent case of β1AR receptor.59,60 However, to investigate a protein landscape, questions arise regarding the relevancy of introducing thermostabilizing residues that tend to freeze proteins, with the resulting improving NMR data at the expense of perturbation of the intrinsic functional dynamics. For instance, in one of the study of β1AR,59 some mutations had to be reversed to observe coupling of the receptor to its cognate G protein. This lack of perdeuteration has several important consequences. First, except two studies using nanodiscs30,44 the vast majority of these studies were carried out in detergent solutions because GPCRs associated with detergent micelles have a faster overall correlation time compared to other media. However, even associated with detergent molecules, a protonated environment precludes the detection of subtle but important variations in the conformational landscape (Figure 2). Moreover, detergents do not form a lipid bilayer around the receptor so they do not mimic very well a native environment, which undoubtedly affects the structure and dynamics of the protein. In particular, some of them display a relative fast chemical exchange at the surface of the receptor that can have an impact on the conformational landscape.65 Moreover, there is a close structural and functional interplay between membrane proteins and lipids, as recently illustrated with BLT2.30 Finally, detergents are known to be a quite destabilizing environment for membrane proteins.66,67 Even though new promising detergents like maltose neopentyl glycol have proved to be useful in the study of various membrane proteins,68 the use of detergents also precludes a detailed analysis of the influence of the membrane structure on receptor dynamics. Indeed, when working for instance with a mix of detergent and lipids, it is still difficult, or even impossible, to tightly control the exact amount of lipids associated with receptor/detergent complexes because of the chemical exchange of detergent molecules that can carry lipids towards free-protein detergent micelles.

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Figure 2. Impact of perdeuteration of GPCRs on the quality of NMR data in solution, part 1: E. coli vs. sf9 expression hosts. Experimental conditions are indicated on the left on each spectrum in addition to the name of the receptor. This includes the level of deuteration, the isotope-labeling scheme of amino acids and their localization (extra- or intra-membrane or both) (highlighted in purple), the use of lipid discs or micelles of detergent (highlighted in red), the temperature and the 1H Larmor frequency of the static magnetic field used (highlighted in green), and the expression host (highlighted in yellow). In panels B to G, the spectrum represented in blue in each inset corresponds to the spectrum displayed in panel A, but at the same scale in both dimensions compared to each spectrum from the literature. The comparison presented herein is only based on published plots of spectra, not on NMR data processing parameters, as for the most part of published spectra presented here the processing parameters were not indicated. (A) 13CH3 methionine region of a perdeuterated and 13CH3-ε labeled Met residues mutant low affinity leukotriene apo BLT2 receptor, that contains methionines in TM α-helices only (Met1,325,349Ala-BLT2), in MSP1D1 nanodiscs. The 2D 1H-13C SOFAST-HMQC TROSY experiment85 has been acquired at 950 MHz 1H Larmor frequency, 298K, and pH 7.4 (adapted with permission from ref. 30. Copyright (2016) American Chemical Society). The dotted lines in blue indicate the position of extracted rows in the 1H dimension displayed in panel H. (B) 13CH3 methionine region of partially deuterated µ-opioid receptor in Lauryl Maltose Neopentyl Glycol (LMNG) detergent micelles (0.08% w/v). The 2D 1H–13C heteronuclear multiple quantum correlation (HMQC) spectrum has been acquired at 800 MHz 1H Larmor frequency, 298K, and pH 7.2 (adapted from ref. 57, Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission). (C) 13CH3 methionine region of partially deuterated β2-adrenergic receptor (containing 14 types of amino acids with a level of deuteration comprised between 80 to 90%) in MSP1 nanodiscs. The 2D 1H–13C HMQC experiment has been acquired at 800 MHz 1H Larmor frequency, 298K, at pH 7.1 (adapted from ref. 44, Copyright Wiley-VCH Verlag GmbH & Co. KGaA. Reproduced with permission). (D) 13CH3 dimethylated lysine (ε-N[13CH3]2-lysines) region of protonated µ-opioid receptor in a mixed micelle detergent/lipid solution (0.01% w/v Maltose Neopentyl Glycol, MNG, associated with 0.001% cholesteryl hemisuccinate, CHS). The 2D 1H–13C HMQC spectrum has been acquired at 700 MHz 1H Larmor frequency (temperature not indicated), at pH 7.4 (adapted from ref. 58 with permission from Nature Publishing Group and Copyright Clearance Center). (E) 13CH3 methionine region of fully protonated β2-adrenergic receptor in β-DDM detergent micelles (0.08% w/v). The 2D 1H–13C SOFAST-HMQC experiment has been acquired at 800 MHz 1H Larmor frequency, 298K, at pH 7.1 (adapted from ref. 53 on courtesy of Nature communications). (F) 13CH3 methionine region of fully protonated thermostabilized (3 thermostabilizing point mutations) β1adrenergic receptor in LMNG detergent micelles (0.04% w/v). The three superimposed 1H,13C HMQC spectra have been acquired at 800 MHz at 288, 298 and 308 K (adapted from ref. 60 on courtesy of Nature communications). (G) 1HN-15N valine region of fully protonated thermostabilized (9 thermostabilizing point mutations) β1-adrenergic receptor (β1AR) in n-decyl-β-D-maltopyranoside detergent micelles (0.1% w/v). The 2D 1H,15N TROSY experiment has been acquired at 800 MHz (or 900 MHz) 1H Larmor frequency, 304K, at pH 7.5. To adjust the scales in the indirect dimension between the spectrum of BLT2 (in blue in the inset) and β 1AR, magnetogyric ratios of 67.262 and −27.116 (106 rad⨯s−1⨯T−1) for respectively 13C and 15N nuclei have been used (spectrum in black adapted from ref. 59 with permission from Nature Publishing Group and Copyright Clearance Center). (H) left, 19F 1D NMR spectrum (experimental spectrum in red, deconvoluted spectra in black dotted lines) of cysteine 265 that carries a fluorinated methyl group in the protonated β2-adrenergic receptor in MNG-3 detergent micelles (0.01% w/v). The experiment was acquired at 298K and pH 7.5. Right, two extracted rows in the 1H dimension from the experiment displayed in A. Magnetogyric ratios of 267.513 and 251.662 (106 rad⨯s−1⨯T−1) for respectively 1H and 19F nuclei have been used to represent all spectra at the same scale (the spectrum on the left is adapted from ref. 56 with permission from Elsevier and Copyright Clearance Center).

Another consequence of the lack of deuteration or perdeuteration is the use of chemically modified amino acids with protonated or fluorinated methyl probes that are used as reporters for NMR detection.51,52,54-56,58 In the case of GPCR studies, it is difficult to gauge the impact on the receptor conformational equilibrium of dimethylated lysines or methylated cysteines but also of the homogeneity of the chemical labeling among a same batch of receptors. Moreover, the approach based on modified amino acids can only deal with the conformational exchange of residues that are solvent-accessible, i.e. not in the transmembrane parts of the receptor. This is due both to the chemical reactions used to modify the lysines or cysteines that usually occur in an aqueous solvent and for a matter of accessibility of the reactants. However, in the case of fluorinated methyl group, despite a lack of resolution because of the use of 1D NMR experiments and an efficient 19F transversal relaxation due to the large chemical shift anisotropy which becomes the major source of line width at high magnetic fields, thus leading to decreased signal to noise ratio and reduced spectra resolution, this nucleus still represents a valuable alternative, in particular to investigate some kinetic barriers in GPCRs (e.g. ref. 56). Indeed, in the case of the observation of fast chemical exchanges at the timescale of the 1H chemical shift, i.e. sharp lines corresponding to an averaging of all the conformations, it could be an advantage of using 19F which covers a wider range of chemical shifts. To be noticed, in some cases, it has been shown in some GPCR studies that 19F chemical shifts were not responsive to conformational changes that are known to occur, based on other experimental methods. This was associated with possible effects of aromatic ring current fields on the chemical shifts of 19F-NMR probes that could hide some expected conformational changes. This has been observed with the β2AR and mammalian rhodopsin, where the 19F-labeling sites, that should have reported conformational changes, were located near aromatic residues.69

NMR STUDIES OF GPCR ENERGY LANDSCAPES WITH PERDEUTERATED RECEPTORS

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To address all the issues above-mentioned, a strategy has been devised that successively associates: overexpression in E. coli which allows the design of appropriate isotope-labeling schemes dedicated to the study of large protein complexes in solution by NMR, and a subsequent in vitro folding followed by a reconstitution of the receptor in nanodiscs, that provides a membrane-like environment and opens the way to the analysis of the impact of the lipid environment on the receptor landscape. This allowed exploration of the conformational ensemble along the TM part of a GPCR through methyl groups of unmodified amino acids in a lipid environment. Central to this strategy is the ability of E. coli to grow in very hostile conditions like 100%-D2O solutions associated to a full incorporation of 13CH3-labeled precursors, leading to the perdeuteration and specific 13CH3-labeling of the receptor that provided an NMR spectral resolution never observed previously in the field for a GPCR embedded in lipid nanodiscs, i.e. below 20 Hz of resolution (Figure 2).30 For a comparison, in nanodiscs of similar size, 10 to 20% of residual protonation around methionines of β2AR have already a dramatic impact on the quality of NMR signals (Figure 2B). Perdeuteration of BLT2 receptor leads also to 13CH3-Met signals narrower than those observed for methionines residues of protonated β2AR in β-DDM micelles (Figure 2C) or the partially deuterated µ-opioid (µOR) receptor in a LMNG detergent solution (Figure 2D). Perdeuteration also leads to similar or even better resolved signals compared to 13CH3-dimethylated protonated lysines located in extra-membrane and mobile parts of µOR in a mixed deter-


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gent/lipid solution (Figure 2E), to narrower linewidths compared to 1HN,15N correlation peaks, especially in the proton dimension (Figure 2F), and to a spectacular difference in the linewidth of signals between rows extracted in the 1H dimension from 2D 1H,13C correlation experiments compared to 19F 1D NMR experiments (Figure 2G). Compared to spectra of GPCRs in detergent solutions expressed in yeast in 100%-D2O media, perdeuterated GPCRs expressed in bacterial systems still provide the finest resolution, allowing the observation of conformational sub-states in lipid nanodiscs (Figure 3).

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Figure 3. Impact of perdeuteration of GPCRs on the quality of NMR data in solution, part 2: E. coli vs. Pichia pastoris expression hosts. Legend same as in Figure 2. Experimental conditions are indicated on the left on each spectrum in addition to the name of the receptor. In panels B and C, the spectrum represented in blue in each inset corresponds to the spectrum displayed in panel A, but at the same scale in both dimensions compared to each spectrum in black or green (from the literature). (A) same as Figure 2A, i.e. 13CH3 methionine region of a perdeuterated and 13CH3-ε labeled Met residues mutant low affinity leukotriene apo BLT2 receptor that contains methionines in TM α-helices only (Met1,325,349Ala-BLT2), in MSP1D1 nanodiscs. The 2D 1H-13C SOFAST-HMQC TROSY experiment85 has been acquired at 950 MHz 1H Larmor frequency, 298K, and pH 7.4 (adapted with permission from ref. 30. Copyright (2016) American Chemical Society). (B) δ1-13CH3 isoleucine region of 90% deuterated A2AR receptor in deuterated β-DDM detergent micelles (0.05% w/v). The 2D 1H–13C HMQC spectrum has been acquired at 800 MHz 1H Larmor frequency, 303K, and pH 7.5 (adapted from ref. 62. Reproduced with permission). (C) 1HN15N glycine region of partially A2AR receptor (70% deuterated) in a mixed micelle detergent/lipid solution (0.025% w/v LMNG associated with 0.00125% w/v CHS). The 2D 1H–15N TROSY correlation spectrum has been acquired at 800 MHz 1H Larmor frequency, 307K, at pH 7.0 (adapted from ref. 63, with permission from Elsevier and Copyright Clearance Center). To adjust the scales in the indirect dimension between the spectrum of BLT2 (in blue in the inset) and A2AR, magnetogyric ratios of 67.262 and −27.116 (106 rad⨯s−1⨯T−1) for respectively 13C and 15N nuclei have been used.

PERSPECTIVES

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Although their number is limited compared to the receptors produced in the most popular sf9 expression system, several bacterially expressed GPCRs have already been used in biophysical studies (fluorescence, solution-and solid-state NMRL). These include the LTB4 receptors BLT1 and BLT2,70 the ghrelin receptor GHSR,37,71 the neuropeptide Y2 receptor NPY2R,72-74 the neurotensin receptor NTS1R,75,76 the cannabinoid receptor CB2R77 or the chemokine receptor CXCR1.78 Among them, many have been obtained after in vitro folding of proteins purified from bacterial inclusion bodies (BLT1, BLT2, GHSR, NPY2R, CXCR1) (for a review, see ref. 10). A striking illustration of the efficiency of the bacterial expression system to the production of labeled GPCRs for NMR studies relies on the production of perdeuterated proteins at 13C natural abundance that contain protonated and 13C-labeled methyl groups. To this end, only E. coli offers the best compromise in term of genetic engineering and protein production yield. Above all, a large catalog of precursors are now available to incorporate 13C-labeled and protonated methyl groups in amino acids, including prochiral labeling in the isopropyl group of Val and Leu residues, or the labeling of only one methyl group in the side chain of Ile for instance. In an energy landscape formalism, applying such labeling strategy opens an avenue to characterize conformational ensem-


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bles, including detection of lowly populated and transiently formed states by spin relaxation studies,6,79-81 and also the use of paramagnetic probes.82-84

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Once the conformational ensemble delineated, the next step in the characterization of GPCR energy landscape is to detect and characterize kinetic barriers separating these different conformations. Solution-state NMR spectroscopy represents a very powerful approach through the use of nuclear spin relaxation phenomena. If present, chemical exchanges contribute to spin relaxation and can be characterized in details by determining the kinetic rates of exchange and populations involved, opening an access to the determination of activation energies to thermodynamics data, providing that experiments can be carried at several temperatures. In certain cases, based on de novo protein structure determination using powerful computational approaches, structural information can be extracted based on variation in NMR chemical shifts observed in chemical exchanges.80 Although the structural determination of large proteins or protein complexes is reserved to X-ray diffraction on 3D crystals or to cryo-electron microscopy, that strategy could be applied to gain structural information in GPCR sub-domains in excited states in complement to low energy, i.e. highly populated and long-lived states, that can be crystallized. One of the main drawbacks of relaxation measurements is that they require quite concentrated samples (at least 100200 µM) to obtain accurate measurements and perform a series of experiments in a realistic experiment time. To this aim, the production of receptors in E. coli IBs at a reasonable cost represents an additional advantage compared to other hosts. Perdeuteration means also a homogeneous deuterated environment around nuclei under investigation, which ensures not only a better signal-tonoise ratio but also a simplification of the interpretation of relaxation data. Again, to that purpose, methyl groups can be advantageously used thanks to their favorable relaxation properties in large proteins or protein complexes. Moreover, to simplify the interpretation of the results, it can be helpful to work with a mono-protonated methyl groups in order to correctly interpret the relaxation experiments by removing intra-dipolar 1H-1H relaxation mechanisms.81 In our hands, using 13CHD2-labeled precursors, as expected, did not change the protein yield, i.e. 4 to 6 mg of pure receptor per liter of culture in 100% D2O solutions can be obtained following a purification of IBs (unpublished data).

SUMMARY AND OUTLOOK E. coli is well-adapted for producing proteins in the context of biophysical studies. This is important in the GPCR field where molecular information besides crystal structures is urgently required to illuminate how signaling behaviors are controlled by the conformational dynamics of the receptors and by their environment (lipids, dimerization partners, signaling proteinsL). The versatility of the bacterial expression system for developing complex isotope-labeling strategies for NMR, that are actually out of reach of more complex expression systems, is particularly relevant as E. coli amino acid metabolic pathways can be carefully tuned to produce relevant isotope-labelings for NMR studies of large proteins, in parallel to its capacity for this organism to grow in 100%-D2O medium. Expressing 13CH3-labeled amino acids at specific residue sites in perdeuterated GPCRs through an approach based on accumulation in IBs allows the production of large protein amounts. Associated with efficient in vitro folding procedure and insertion into lipid nanodiscs and, eventually, subsequent purification of active fractions, this opens the way to state-of-the-art NMR techniques to investigate the dynamics of these receptors.

AUTHOR INFORMATION Corresponding Authors

350 *[email protected] *[email protected]

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360

ORCID Marina Casiraghi: Marjorie Damian: Ewen Lescop: Jean-Louis Banères:

Laurent J. Catoire:

0000-0003-0627-2288 0000-0002-9163-2673 0000-0002-2623-9365 0000-0001-7078-1285 0000-0001-6839-3312

ACKNOWLEDGMENT

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This work was supported by grants from the Centre National de la Recherche Scientifique, Universités de Montpellier and Paris Diderot, Agence Nationale de la Recherche (ANR-13-BSV8-0006-01 and ANR-17-CE11-0011), Laboratoire d’Excellence (LabEx) DYNA-


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MO and Equipements d’Excellence (EQUIPEX) CACSICE from the French Ministry of Research. Financial support from the TGIRRMN-THC Fr3050 CNRS for conducting the research is also gratefully acknowledged.

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Okude, J.; Ueda, T.; Kofuku, Y.; Sato, M.; Nobuyama, N.; Kondo, K.; Shiraishi, Y.; Mizumura, T.; Onishi, K.; Natsume, M.; Maeda, M.; Tsujishita, H.; Kuranaga, T.; Inoue, M.; Shimada, I. Identification of a Conformational Equilibrium That Determines the Efficacy and Functional Selectivity of the µ-Opioid Receptor. Angew. Chem. Int. Ed. Engl. 2015, 54, 15771. Sounier, R.; Mas, C.; Steyaert, J.; Laeremans, T.; Manglik, A.; Huang, W.; Kobilka, B. K.; Demene, H.; Granier, S. Propagation of conformational changes during µ-opioid receptor activation. Nature 2015, 524, 375. Isogai, S.; Deupi, X.; Opitz, C.; Heydenreich, F. M.; Tsai, C. J.; Brueckner, F.; Schertler, G. F.; Veprintsev, D. B.; Grzesiek, S. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor. Nature 2016, 530, 237. Solt, A. S.; Bostock, M. J.; Shrestha, B.; Kumar, P.; Warne, T.; Tate, C. G.; Nietlispach, D. 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555 TOC graphic:

560

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Table 1. Different approaches for the study of β2AR, μOR, β1AR, A2AR and BLT2 conformational ensembles by solution-state NMR spectroscopy. Receptor

Modifications

Type of isotope labeling

Expression system

Environment

NMR experiment

2AR51

Truncation after Gly365

(13CH3)2-Lys reductive

sf9 insect cells

Detergent

2D 1H,13C

di-methylation 2AR52

Thermal stabilizing mutation E122W, C-term truncation at residue 348,

13

C19F3-Cys covalently

(-DDM) sf9 insect cells

labeled

Detergent

1 D 19F NMR

(-DDM, +CHS)


Detergent

2D 1H,13C

245-259 (ECL3) 2AR53


-13CH3-Met

sf9 insect cells

(-DDM) 2AR54

2AR-4 Cys deletion

13

C19F3-Cys265

sf9 insect cells

Detergent (MNG-3)

1 D 19F NMR +Lorentzian deconvolution

2AR55

2AR44

Deletion of the 5

-13CH3-Met

extra-membraneMet

(3 Met retained)


-13CH13-Met

sf9 insect cells

μOR57

2AR-4 Cys deletion

F158W, 6 extra-membrane Met

13

C19F3-Cys265

-13CH3-Met

2D 1H,13C

(-DDM) Sf9 insect cells

(5 Met retained) 2AR56

Detergent

Lipid nanodiscs

2D 1H,13C

(POPC + POPG) sf9 insect cells

sf9 insect cells

Detergent

1D 19F NMR

(MNG-3)


Detergent

2D 1H,13C

(LMNG) μOR58

M72T mutation

(13CH3)2-Lys reductive

sf9 insect cells

di-methylation 1AR59


15

N-Val

Detergent

2D 1H,13C

(MNG-3, +CHS) sf9 insect cells

3 point mutations, N-term & ICL3

Detergent

2D 1H,15N

(DM)

truncations A2AR61

AR60

V229C mutation


13

C19F3 -Cys229

-13CH3-Met

Yeast, Pichia pastoris Detergent

Sf9 insect cells

2 point mutations A2AR62

N154Q mutation

BLT230

N154Q mutation

(MNG-3)


Detergent

2D 1H,13C

(LMNG) -13CH3-Ile


A2AR63

1D 19F NMR

uniformly 15N-labeled

( H--DDM)


(LMNG, +CHS)

Native sequence or mutant with

-13CH3-Met and

E. coli in

Lipid nanodiscs

3 extra-membrane Met mutations

-13CH3-Ile

100% D2O

(DMPC + CHS)


2D 1H,13C

2

2D 1H,15N

2D 1H,13C

SOLUBILIZATION & IN VITRO TRANSFER TO 13CH3,u-2H GPCR BACTERIAL FOLDING PageEXPRESSION 15 of 18 PURIFICATION IN BiochemistryLIPID NANODISCS DENATURING CONDITIONS + AMPHIPHILE TO INCLUSION BODIES + LIPOPROTEINS [detergents, amphipols, lipids] & LIPIDS + SDS + 13CH3,u-2H,12C 1 precursors

2 3 4 5 6 7 8

SOLUTION-STATE NMR

16 Hz

22 Hz

13CH3 200 Hz

INSTANTANEOUS SDS REMOVAL BY K+ IONS

100%-D2O

AFFINITY

CHROMATOGRAPHIES ACS Paragon Plus Environment pure two-secondary GPCR α-helical content

TO METALS AND/OR LIGANDS

non-100% active GPCR

100% active & stable GPCR

2.4

2.0 1.6 [ppm] 1H

A

Biochemistry

1H [ppm]

1.98

D

17.0

18.0

expression host: insect cells (sf9)

2.0

1.8

1.8

1H [ppm]

1.6

1.4

F

105 110

BLT2

protonated ε- (13 CH3 )2 -Lys (extra-membrane a.a.) mixed lipid/detergent solution (MNG + CHS) temperature not indicated 700 MHz (νH) expression host: insect cells (sf9) β1AR (3 thermostabilizing mutations) protonated ε- 13CH3 -Met (trans & extra-membrane a.a.) detergent solution (LMNG) 15 to 35°C 800 MHz (νH)

45 46 47

BLT2

3.1

3.0

2.9

125 9.0

8.5

8.0

1H [ppm]

2.8

2.7

2.6

1H [ppm]

16.0 17.0

BLT2

18.0 19.0 2.2

2.0

1.8

1.4

1.6

1H [ppm] 22 Hz

16 Hz

BLT2

β2AR protonated 13 CF3 -Cys (extra-membrane a.a.) detergent solution (MNG-3) 25°C 600 MHz (νH) 7.5 7.0 expression host: ACS Paragon Plus Environment insect cells (sf9)

200 Hz

BLT2

H

120

9.5

1.2

44

1H [ppm]

115

1.4

1.6

1H [ppm]


15N [ppm]


2.0

BLT2

expression host: insect cells (sf9) β1AR (9 thermostabilizing mutations) protonated 15 -Val N (trans & extra-membrane a.a.) detergent solution (DM) 31°C 800 or 900 MHz (νH)

17.5

2.2

13C [ppm]

β2 AR protonated ε- 13CH3 -Met (transmembrane a.a.) detergent solution (βDDM) 25°C 800 MHz (νH)

17.0

18.0

μ OR

2.2

BLT2

16.5

1.96

BLT2

2.4

partially deuterated (8 amino acids deuterated) ε- 13CH3 -Met (transmembrane a.a.) detergent solution (LMNG) 25°C 800 MHz (νH) expression host: insect cells (sf9)

13C [ppm]

β2 AR partially deuterated (14 amino acids deuterated) (50 to 90% 2H incorporation rate) ε- 13CH3 -Met (transmembrane a.a.) lipid discs (lipoprotein: MSP1) 25°C 800 MHz (νH)

2.00

13C [ppm]

G

2.02

13C [ppm]

E

B

14.8

expression host: E. coli

C

13C [ppm]

14.4

M197

μ OR

13C [ppm]

BLT2 perdeuterated ε- 13CH3 -Met (transmembrane a.a.) lipid discs (lipoprotein: MSP1D1) 25°C 950 MHz (νH)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

14.0

M105

Page 16 of 18

16 Hz 200 Hz

-60.0

-61.0

-62.0

200 Hz

2.4

2.0

1H [ppm]

1.6

Page 17 of 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38

Biochemistry


200 Biochemistry Page 18 ofHz 18

1 2 3 4 5 6 CS7Paragon Plus Environmen 8 2.0 1.6 2.4 9 1H [ppm]

Illuminating the energy landscape of GPCRs: the key contribution of

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