Structure of the Third Intracellular Loop of the Vasopressin V2 Receptor and Conformational Changes upon Binding to gC1qR

J. Mol. Biol. (2009) 388, 491–507

doi:10.1016/j.jmb.2009.02.065

Available online at www.sciencedirect.com

Structure of the Third Intracellular Loop of the Vasopressin V2 Receptor and Conformational Changes upon Binding to gC1qR Gaëtan Bellot 1,2 , Sébastien Granier 3,4 , William Bourguet 1,2 , René Seyer 3,4 , Rita Rahmeh 3,4 , Bernard Mouillac 3,4 , Robert Pascal 5 , Christiane Mendre 3,4 and Hélène Déméné 1,2 ⁎ 1

INSERM U554, F34090 Montpellier, France 2

CNRS UMR5048, Université Montpellier 1 et 2, Centre de Biochimie Structurale, F34090 Montpellier, France 3

INSERM U661, F34094 Montpellier, France 4

CNRS UMR5203, Université Montpellier 1 et 2, Institut de Génomique Fonctionnelle, F34094 Montpellier, France 5

UMR5247, CNRS, Université Montpellier 1 et 2, Institut des Biomolécules Max Mousseron (IBMM), F34095 Montpellier, France Received 4 August 2008; received in revised form 23 December 2008; accepted 27 February 2009 Available online 12 March 2009

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GαS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted αhelical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GαS. Moreover, we could estimate the apparent Kd of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist. © 2009 Elsevier Ltd. All rights reserved.

Edited by A. G. Palmer III

Keywords: V2 vasopressin receptor; NMR structure; i3 loop; gC1qR; complex

*Corresponding author. E-mail address: [email protected]. Abbreviations used: DMSO, dimethyl sulfoxide; DPC, dodecyl phosphocholine; Gd(DTPA-BMA), Gd-diethylenetriamine pentaacetic acid–bismethylamide; GPCR, G-protein-coupled receptor; gC1qR, receptor for the globular domains of the C1q complement; HSQC, heteronuclear single quantum coherence; NOE, nuclear Overhauser effect; PDB, Protein Data Bank; RCI, random coil index; RDC, residual dipolar coupling; STD, saturation transfer difference; TALOS, torsion angle likelihood obtained from shift and sequence similarity; TM, transmembrane; TOCSY, total correlated spectroscopy; TR-NOESY, transferred nuclear Overhauser effect spectroscopy. 0022-2836/$ - see front matter © 2009 Elsevier Ltd. All rights reserved.

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Introduction G-protein-coupled receptors (GPCRs)1 are the most abundant transmembrane (TM) proteins expressed at the surface of cells. They are dedicated to the behavior adaptation of the cell in responses to the stimuli from other cells and from the environment. All GPCRs share a common architecture of seven TM helices. Association of GPCRs with their specific ligands at the extracellular interface triggers conformational changes in the TM and subsequently in cytoplasmic domains, which enables them to interact with heterotrimeric G proteins and initiate the transduction cascade.1 GPCRs are now recognized to interact with many other proteins called GPCR-interacting proteins. These proteins are implicated in GPCR targeting to and from the plasma membrane and in the finetuning of their signaling properties.2 Despite the crucial role played by GPCRs and GPCR-interacting proteins, many of the processes of signal transduction by GPCRs are not well understood at the molecular level. This can be partly attributed to the lack of structural information about GPCRs' cytoplasmic interface and in particular about the i3 interface. There are now five elucidated structures of eukaryotic GPCRs: liganded bovine rhodopsin,3–8 unliganded bovine opsin,9 β2-adrenergic receptor,10–12 β1-adrenergic receptor,13 and squid rhodopsin.14,15 The electronic densities of the third intracellular loops are, however, missing in most structures (including the β1- and β2-adrenergic receptor), and the conformational variability of the cytoplasmic loops, if present, is large among the elucidated structures. Moreover, within the GPCR family, the intracellular loops share very few conserved residues,16 which is particularly the case for the i3 loop whose length is of considerable variability.17 To what degree these crystalline structures represent a structural paradigm for other GPCRs at the cytoplasmic interface is thus debatable, especially at the i3 loop level. Considering the low amount of available structural data on entire receptors, spectroscopic studies of synthetic peptides consisting of receptor fragments provided an insightful method for the identification of the structural and functional features of the intracellular loops of GPCRs, and this method has been widely used.18–28 The V2 vasopressin receptor belongs to the class A of GPCRs. It activates the GαS protein via its third intracellular loop i329,30 and part of its proximal Cterminal region31 to produce its renal antidiuretic effect.29 The third intracellular loop is 50 amino acids long, a sequence length that makes its tridimensional structure not amenable to ab initio modeling. The synthetic peptide inhibits GTP binding by the GαS protein.20 Using a cyclized form, named thereafter i3_cyc, as a bait in a proteomic approach led to the identification of the receptor of the C1q complement (gC1qR) as an interacting protein.32,33 gC1qR is a multifunctional and multicompartmental cellular protein.34 It is also known as the p33/p32 protein or as hyaluronan-binding protein 1. It is folded as a trimer arranged in a unique “doughnut”-like disk,

with a neutral face and a highly acidic one.35 Depending on its ligand-binding state, it plays various roles in different cellular processes such as cell adhesion, inflammation, immune response, or tumor invasion. Coupling of gC1qR with GPCRs has been only recently brought to light.33,36–38 The functional link still remains elusive today, but a chaperone-like activity has been suggested for GPCRs and for other membrane proteins as well.37–41 To get an insight into the structure–activity relationship of the V2 i3 loop, we have undertaken a study of the structure of i3_cyc isolated and within its complex with gC1qR. Our study shows that i3_cyc folded as a well-defined lefttwisted α-helical hairpin. The linker connecting both hairpin helices was constituted by an arginine cluster and was more flexible than the rest of the molecule. Comparison with the available rhodopsin structures yielded three different (two bovine and one squid) rhodopsin structures where the i3 loops present a similar fold despite differences in lengths and twist angles.5,14 Building of a receptor model based on i3_cyc and rhodopsin structures shed light on several features such as the side-chain orientation of heavily charged residues. In the V2 model, the length and particular twist of the i3 hairpin resulted in an original structural cytoplasmic motif, which may be relevant for the coupling with the GαS protein. When investigating the i3cyc/gC1qR complex, we determined the apparent Kd of the interaction from fluorescence anisotropy experiments. However, precipitation problems forced us to work with a shorter and more soluble form of the peptide, i3_short. NMR saturation transfer difference (STD) measurements confirmed that the interaction surface with gC1qR was the arginine cluster as inferred from previous mutagenesis experiments.38 Transferred nuclear Overhauser effect spectroscopy (TR-NOESY) experiments revealed that gC1qR binding induced the folding of the otherwise disordered i3_short peptide into a spiral path. Introduction of the gC1qR-bound nuclear Overhauser effect (NOE) constraints (observed for the i3_short peptide) into calculations for the entire i3_cyc loop suggested that gC1qR binding would rigidify the central arginine cluster and might change the packing angle of the cytosolic hairpin helices and of their TM continuations, TM V and TM VI. To evaluate the presence of such a conformational rearrangement in the context of a full-length receptor, we developed an intrinsic tryptophan fluorescence assay using the entire purified V2 receptor. Both the NMR calculations and the fluorescence assays support the presence of structural changes in the i3 loop upon gC1qR binding, which propagate to the TM domain.

Results NMR structural parameters of i3_cyc in DPC micelles A previous study has shown that the i3_cyc structure is random coil in solution but possesses a significant amount of helices in dodecyl phosphocholine

NMR Structure of the GPCR V2 i3 Loop

493

(DPC) micelles.20 We further studied the structure of i3_cyc in the presence of deuterated DPC-d38 by 1H and 13C NMR at 600 and 800 MHz (Fig. 1b). The resonances are concentrated in a narrow frequency window, which is not surprising given the high amount of helices expected (N 50%). The NOE pattern of sequential and medium-range NOEs (Fig. 1c) clearly established the presence of a helix in the Nterminal segment (residues 225–243), shortly interrupted at the level of Pro238, and of a long helix in the C-terminal part extending from Gly254 to Arg271. The central part of the loop, delimited by Gly245 and Gly250, appeared more disordered on the basis of i/i + 3 NOE connectivities.

presence of Gd-diethylenetriamine pentaacetic acid– bismethylamide [Gd(DTPA-BMA)]. 45 Figure 2b depicts the minimized mean structure of i3_cyc colored as a function of the calculated attenuation factor. The residues most exposed to solvent were found to locate around and within the basic cluster. Hence, the structure of i3_cyc could be characterized as a well-defined hairpin presenting a highly positively charged linker. Moreover, the visualization of the i3_cyc structure and attenuation factors showed that some hydrophilic residues, namely, Gln225, Arg230, Glu231, Glu242, Glu258, Lys268, and Arg271, were apparently orienting their side chain towards the lipid chains.

NMR-derived 3D structure of i3_cyc in DPC micelles

Comparison between the structures of i3_cyc and of the available i3 loops of rhodopsin

The collected NOEs and chemical shifts were introduced as constraints in structure calculations. The superposition of the 20 best structures calculated by Aria 2.0 is shown in Fig. 2a, and the structural and energetic statistics are listed in Table 1. i3_cyc folded as a tightly closed and left-twisted α-helical hairpin. The N-terminal helix spanned the Val226-Leu236 residues and was followed by a short helix turn in the Pro238-Ser241 segment. A long bended Cterminal helix extended from Gly250 to Val270. The hairpin helices could be superposed simultaneously (Gln225-Glu242 and Gly254-Thr273 segments) with an r.m.s.d. value of 0.5 ± 0.2 Å for the backbone atoms. Their packing angle was 47 ± 2° and was, hence, remarkably defined. It was stabilized by numerous hydrophobic interactions, between the side chains of Ile228 and Val270, Ile228 and Met266, Phe229 and Val270, Glu231 and Ser263, Ser235 and Ser263, and Pro238 and Ser263. Omission of the 10 interhelix NOEs in structure calculations led to a “breathing” of the hairpin helices, with no particular orientation left. The linker between N- and C-terminal helices was constituted by the arginine cluster flanked by the glycines Gly245 and Gly254. It was formed from a succession of I and IV turns and was more disordered than the rest of the molecule, with an r.m.s.d. value of 1.2 ± 0.4 Å. The overlap of the aliphatic resonances within the four arginine residues could have, however, easily misled to the assignment of medium-range connectivities as sequential ones, resulting in locally less constrained structures. To clear up this point, we analyzed the chemical shifts in flexibility terms through the dedicated web server of Berjanskii and Wishart.42–44 Random coil index (RCI) analysis (Fig. 2b) proved that the central part of the i3_cyc loop was actually more flexible. Nevertheless, the side-chain distribution of the arginine cluster was rather well defined (Fig. 2c), with Arg243, Arg247, Arg248, and Arg251 presenting their side chain pointing to the same side of i3_cyc as opposed to Arg249 and Arg252 (with the Arg246 side chain being equally populated between both faces). To determine the solvent exposure of the arginine cluster, we conducted NMR experiments in the

Our previous NMR studies had revealed an interesting similarity between the i2 intracellular loops of the V1a vasopressin receptor and the bovine rhodopsin receptor.23 The structure of i3 is variable between the different crystal structures because it is at the interface of crystal contacts. We found three GPCR crystal structures that presented a similar fold: liganded bovine rhodopsin (1GZM), unliganded bovine opsin (3CAP), and liganded squid rhodopsin (1E12). In these structures, the i3 loops of bovine (30 amino acids) and squid rhodopsin (45 amino acids) fold as left-twisted α-helical hairpins as well, characterized by packing angles of 29°, 13°, and 22° for liganded bovine rhodopsin, free bovine opsin, and liganded squid rhodopsin, respectively. The i3 loop of squid rhodopsin14,15 is particularly interesting to compare with, since it is only five residues shorter than i3_cyc. The original feature of this structure is that the N- and C-terminal helices, each 19 amino acids long, extend considerably the TM V and TM VI helices, forming a rigid, long cytoplasmic protrusion. The main difference between i3_cyc and the squid i3 loop was the packing angle value of 22° and 44°, respectively. However, the twist was identical (i.e., left) between these three α-helical hairpin structures, suggesting that the i3_cyc structure could be easily grafted onto the rhodopsin structures without significant distortion of TM V and TM VI helices. Model of the V2 receptor Using the Modeller software, we built a model of the V2 receptor based on the structure of bovine rhodopsin (1GZM) and the NMR-calculated structure of i3_cyc. This structure was chosen because the packing angle of its i3 loop was the closest to i3_cyc. A model was also constructed based on the structure of the β2-adrenergic receptor, but as the architectures of TM helices in both receptors are very close, differences were not significant. As expected, grafting of the NMR i3_cyc structure onto the rhodopsin structure could produce a model of the V2 receptor without high distortion of the original templates (Fig. 2c and d). Superposition of backbone atoms of TM V and TM VI of the V2 model onto the rhodopsin structure

494

NMR Structure of the GPCR V2 i3 Loop

Fig. 1. NMR spectroscopic and structural parameters of the third intracellular loop of the V2 receptor (i3_cyc). (a) Primary sequences of i3_cyc (third intracellular loop of the V2 receptor), i3_short (central portion of i3_cyc), and ETA i3 (third intracellular loop of the endothelin type A receptor). (b) TOCSY fingerprint of i3_cyc recorded at 800 MHz in the presence of 100 equivalents of DPC (pH 4). HN-Hα correlation cross peaks are annotated according to the residue number in the sequence. (c) Summary of sequential and medium-range NOEs (collected with a mixing time of 100 ms) characterizing the elements of secondary structure of i3_cyc. The sequence of i3_cyc is depicted as head. The gray box corresponds to the sequence of i3_short.

yielded an r.m.s.d. of 1.2 Å, reflecting a limited change of TM orientations in order to accommodate for the higher packing angle of i3_cyc loop. On the other hand, backbone atom superposition of the NMR i3_cyc structure onto the i3 loop of the V2 model yielded an r.m.s.d. of 0.4 Å. In the model, the N- and

C-terminal helices of the hairpin loop represented the cytosolic helix extensions of the TM V and TM VI helices (Fig. 2d). We further reexamined the position of hydrophilic residues, which were found to face the micelle environment in the isolated i3_cyc structure, within

NMR Structure of the GPCR V2 i3 Loop

495

Fig. 2. Structure of the V2 intracellular loop i3_cyc either isolated or integrated in the whole receptor. Superposition of the 20 best structures of i3_cyc. (a) Superposition was done using the backbone atoms of segments Gln225-Glu242 and Gly254-Thr273. (b) Mean minimized structure of the backbone of i3_cyc. The width of the backbone line is proportional to the flexibility as calculated by the RCI method.43 Color of the backbone depends on the intensity of attenuation factor in the presence of Gd(DTPA-BMA): red corresponds to the most exposed residues and blue corresponds to the most protected residues. (c) Model of the V2 receptor constructed with the bovine structure (PDB code: 1GZM) and the NMRderived structure of i3_cyc as templates. The side chains of strongly polar and charged amino acids of the i3 loop are represented. Roman numerals refer to TM domains. For clarity, the first 25 and last 25 amino acids of the receptor are not represented since they were generated in extended conformations as they were constraint free. (d) Cytoplasmic view of the squid rhodopsin structure and of the V2 model.

the frame of the whole receptor model (Fig. 2c). If one extrapolates the results obtained from docking the bovine opsin structure into the cryo-electronic microscopy map,5,46 the cytoplasmic part protruding above the lipid head bilayer would be formed by the Glu231Ala267 segment (corresponding to the Thr229-Lys248 opsin segment). In the whole receptor model, the Gln225 side chain in TM V would interact with TM VI. The side-chain orientations of Arg230 and Glu231 suggested an interaction with the N-terminal extremity of the i2 loop. Lys268 and Arg271 side chains (TM VI) were likely to interact with the C-terminal of TM VII or cytoplasmic proteins. In addition, Arg230

and Lys268 represented positively charged residues at the boundaries of the phospholipid bilayer and, thus, would potentially interact with the negatively charged polar heads. The side chain of the other charged residues, Glu242, Glu258, and those of the arginine cluster Arg243-Arg252, would be now fully exposed to solvent and might interact with cytoplasmic proteins. Biophysical studies of the i3_cyc/gC1qR complex We resorted to various methods to characterize the structure of the i3_cyc/gC1qR complex, but the study

NMR Structure of the GPCR V2 i3 Loop

496 Table 1 No. of experimental distance restraints Intraresidue Sequential (|i − j| = 1) Medium range (1 b |i − j| ≤ 4) Long range (|i − j| N 4) Total Hydrogen bonds Dihedral angle Dihedral angle restraints Number N5° Maximum distance constraint violation (Å) NOE distance violations Number N0.3 Å Maximum violation (Å) Deviations from ideal covalent geometry Bond lengths (Å) Bond angles (°) Impropers (°) Average r.m.s.d. (Å) Backbone atoms (all residues) Heavy atoms (all residues) Backbone atoms (residues 225–242 and 254–273) Heavy atoms (residues 225–242 and 254–273) Ramachandran analysis (%) Most favored Additional allowed Generously allowed Disallowed

469 199 141 17 826 22 40 0.85 ± 0.22 1.41 ± 0.07 1.4 0.07 ± 0.01 0.0073 ± 0.0002 1.9358 ± 0.0053 1.1204 ± 0.0103 0.91 ± 0.29 1.87 ± 0.50 0.45 ± 0.16

To get qualitative insight into the gC1qR/i3_cyc complex, we also performed limited proteolysis experiments on gC1qR, in the presence and absence of i3_cyc. Chymotrypsin was chosen as it preferentially cuts after phenylalanine, tyrosine, and tryptophan residues, which are mostly located on the acidic face of gC1qR. Figure 3b depicts the SDS-PAGE analysis of the digests. gC1qR has a theoretical molecular mass of 23 kDa but migrates in SDSPAGE gels with an apparent higher molecular weight as a result of its highly acidic pI (4.2).35 As visible on Fig. 3b, the addition of the i3_cyc loop considerably slowed down the digestion process. The digestion delay was dose dependent (data not shown). These results confirmed the interaction and additionally suggested that the interaction surface of gC1qR was its acidic face. As a control, we used a peptide mimicking the i3 intracellular loop of the ETA receptor (Fig. 3b, lane 5), which also possesses a very basic pI (10 versus 11). The digestion profile of gC1qR incubated with various amounts of the ETA endothelin

0.88 ± 0.24 80.3 16.3 3.4 0.0

has been hampered by serious problems of precipitation. Previous biophysical studies had shown that gC1qR adopts a compact globular shape in a large range of pH, but only in salt concentrations above 150 mM.47 On the other hand, i3_cyc tended to aggregate above pH 5, and the presence of salt promoted its progressive precipitation at all pH. Despite this, we systematically investigated the solubility of the i3_cyc/gC1qR complex in a wide range of pH (pH 3–11), using an extensive set of salts following the Hofmeister series and monitoring the effect of various detergents.48 The i3_cyc/ gC1qR complex was stabilized by high concentrations of kosmotropic salts, with the highest solubility observed for 600–800 mM Na2SO4 in the presence of mild detergents. However, even in these conditions, the complex would still slowly precipitate at millimolar and submillimolar concentrations. To circumvent this problem, we labeled i3_cyc with the fluorophore Alexa-488 on its unique primary amine group, the side chain of Lys268. The purity of labeled i3_cyc was assessed by HPLC and estimated to be 99%. Its structure was confirmed by matrix-assisted laser desorption/ionization mass spectrometry (M + H+exp = 6015.88; M + H+theo = 6015). Having Alexa-488-labeled i3_cyc enabled the monitoring of complex formation at nanomolar and micromolar concentrations. Binding of the labeled i3_cyc to gC1qR caused a rise in the fluorophore anisotropy due to the increase in overall tumbling correlation time. Fitting of the titration curve (Fig. 3a) yielded a Kd of 1.8± 0.2 × 10− 6 M.

Fig. 3. Biophysical and biochemical data for the interaction between gC1qR and i3_cyc and i3_short. (a) Increase in fluorescence anisotropy on titration of gC1qR into a solution of Alexa-488–i3_cyc (c = 2 nM). Fitting of the data yielded an apparent Kd of 1.8 ± 0.2 μM. (b) Limited proteolysis (20 min at pH 7 and 20 °C) of gC1qR upon binding to i3_cyc and i3_short visualized by SDS-PAGE chromatography. In the presence of i3_cyc and i3_short, but not the ETA i3, the digestion of gC1qR by chymotrypsin is delayed.

NMR Structure of the GPCR V2 i3 Loop

receptor i3 loop was identical with that of gC1qR alone. This finding confirmed that the in vitro interaction between gC1qR and i3_cyc visualized by gel chromatography was not an artifact due to the presence of the basic peptide by itself. In a previous work, we established that the simultaneous mutation of the six Arg residues of the G250RRRRGRR257 sequence abolishes the interaction with gC1qR. Furthermore, a shorter peptide, i3_short, corresponding to the central portion of i3_cyc (Fig. 1a) and encompassing this arginine cluster, was shown to compete with i3_cyc in the complex formation.38 Interestingly, the proteolysis profile of gC1qR incubated with i3_short (Fig. 3b, lane 4) was identical with the digestion profile obtained with the entire i3_cyc loop. Hence, we restricted the study of the gC1qR/ i3_cyc complex to the study of the gC1qR/i3_short complex. Interaction between i3_short and gC1qR as seen by transferred NOE and STD experiments To provide additional information regarding i3_short binding mode, we performed STD NMR experiments.49,50 The STD NMR technique is a powerful method of epitope mapping by NMR spectroscopy. Resonances of gC1qR were selectively saturated and protons of i3_short that were in close contact with gC1qR could be identified from the STD NMR spectrum. Investigation of the dependence of the STD effect on the irradiation time showed that an irradiation time of 2 s was sufficient to observe a quantifiable STD effect. Because spin diffusion within the proton network of the ligand occurs, the different signal intensities of the different individual protons are best analyzed from introducing the STD amplification factor (see Materials and Methods).49 Figure 4 depicts the dependence of STD amplification factor of i3_short individual protons as a function of the peptide concentration. Such curves theoretically enable the discrimination between protons of the ligand that are in close contact with the target protein and other protons.49 On this basis, the segment corresponding to the arginine cluster and to the beginning of the C-terminal helix of the i3_cyc hairpin (Arg246-Thr253) appeared to be directly in contact with gC1qR.

497 To investigate the peptide-bound conformation, we performed TR-NOESY on the gC1qR/i3_short complexes. The resonances of free and bound i3_short peptides were in fast exchange on the cross-relaxation timescale; hence, the observed TR-NOESY intensities were sums of the NOE intensities in the free and bound states. As the mixing times were short, NOEs of the free peptide represented small uncorrected NOEs compared to NOEs of the bound state. As seen in Fig. 5a, addition of gC1qR promoted the appearance of numerous NH/NH intramolecular NOEs for i3_short (the resonances of the free peptide are listed in Table S2). In addition to NH/NH dipolar correlations, several i/i + 2 and i/i + 3 NOEs appeared as well, suggesting the folding of the i3_short upon binding to gC1qR. A summary of sequential and medium-range TR-NOEs is presented in Fig. 5b. The i3_short structures calculated with these NOEs are shown in Fig. 6a. Structural statistics are listed in Table 2. The position of backbone atoms was reasonably well defined by the TR-NOE restraints. The structures could be superposed with a root-mean-square deviation (r.m.s.d.) of 2.0 ± 0.7 Å for backbone atoms of residues Arg243-Gly254. The gC1qR-bound structure of i3_short was characterized by a succession of I and IV turns; thus, the peptide adopted a U-shaped structure. Comparison of the i3_short structure with the corresponding segment of the DPC-bound i3_cyc structure showed that the localization of turns was different, in line with the different localizations of i/i + 2 NOEs. gC1qR-bound i3_short adopted a more extended structure due to the presence of successive turns at positions 18, 20, 21, 24, 25, and 27 (versus 18, 21, 22, and 26 for DPC-bound i3_cyc). Superposition of the 10 structures of i3_short onto the best structure of i3_cyc (backbone atoms of residues Glu242-Gly254) yielded an average r.m.s.d. of 3.7 ± 0.3 Å (Fig. 6b). We thus launched calculations to simulate the effect of gC1qR binding for the whole i3_cyc structure. Simulation of gC1qR binding effect on the i3_cyc structure We introduced the TR-NOEs of the i3_short/gC1qR complex in the constraints list of isolated i3_cyc and suppressed the corresponding original NOEs and the TALOS (torsion angle likelihood obtained from shift

Fig. 4. Interaction surface of i3_short with gC1qR. The STD amplification factors Si were calculated with Si = (I0 − Isat)/I0 × ligand excess. Theory predicts that the higher the amplification factor, the closer to gC1qR the residue is.

498

NMR Structure of the GPCR V2 i3 Loop

Fig. 5. Folding of i3_short induced by gC1qR binding as detected by TR-NOESY. (a) Comparison of the region of the NH/NH dipolar correlation peaks for the NOESY experiments (mixing time, 300 ms) recorded for i3_short (c = 4 mM) alone (left) and in the presence of 50 μM gC1qR (right). (b) Summary of sequential and medium-range NOEs (collected with a mixing time of 300 ms) characterizing the elements of the secondary structure of i3_short bound to gC1qR (i3_short bound).

and sequence similarity) constraints for this segment. Compared with the structure of isolated i3_cyc, the arginine-rich hairpin linker was, as expected, more defined and more extended due to the presence of successive turns at positions 18, 20, 21, 24, 25, and 26. More interestingly, as a result of this local folding of its linker, the hairpin loop experienced a significant closing upon binding with the twist angle changed from 47° to 32° (Fig. S3). However, as 4 of the original 17 interhelix NOEs were violated, this change in angle only suggests that the orientation and/or conformation of the hairpin helices in isolated i3cyc is not compatible with the structure of gC1q-R-bound i3_short. To further investigate this phenomenon, we have recalculated the “gC1qR-bound” structure of i3_cyc, but we froze the “native” orientation of i3_cyc helices by applying artificial residual dipolar coupling (RDC) constants on them. In the resulting structures, despite the addition of structural constraints, there was an increase in structural disorder, with the r.m.s. d. increasing from 0.4 ± 0.2 to 0.9 ± 0.2 and from 0.9 ± 0.4 to 1.9 ± 0.6 for the helices and hairpin linker, respectively (Fig. S4). This increase in structural disorder, in particular at the level of the hairpin

helices, was retrieved even if the N- and C-terminal residues of i3_short (Ser241 and Ser255) were left free of NOE constraints in the simulations. In our experience, such elevation of conformational flexibility as the number of structural constraints increases directly reflects the incompatibility of the different NMR constraint sets.51 Influence of gC1qR on binding curves to SR121463 antagonist by the purified recombinant V2 receptor We had previously shown that the purified recombinant V2 receptor binds to gC1qR.38 To provide some clues about the existence of a gC1qRinduced conformational change in the full-length receptor, we developed an intrinsic tryptophan fluorescence assay using purified V2. Addition of SR121463, a nonpeptidic nonfluorescent antagonist, to V2 led to a partial quenching of the fluorescence of its tryptophan residues. Figure 7a shows the binding curve as a function of the added SR121463 concentration. The first part of the curve was sigmoid, but no plateau was reached at higher concentrations of

NMR Structure of the GPCR V2 i3 Loop

499

Fig. 6. i3_short structures calculated using the i3_short-bound NMR TR-NOEs. (a) Superposition of the 10 best structures calculated for gC1qR-bound i3_short. Superposition was done taking into account backbone atoms of the Glu242-Gly254. For clarity, the backbone chains are represented as ribbons. (b) Superposition of the 10 best i3_short structures onto a representative micelle-bound i3_cyc structure. Superposition was done taking into account backbone atoms of the Glu243-Gly254 segments. The i3_cyc structure is represented using a ribbon representation (gray), whereas the i3_short structures (black) are represented by Cα traces.

antagonist, maybe due to deleterious effects of dimethyl sulfoxide (DMSO), the solvent of SR121463. The exact determination of the antagonist dissociation constant (Kd) was thus not possible, but the data were, however, compatible with a Kd in the micromolar range. As a negative control, we used the V2 receptor, which was refolded in sodium dodecyl sulfate micelles (V2-SDS) and adopted a nonnative structure. V2-SDS exhibited no fluorescence response to SR121463 (Fig. 7b). As for the correctly refolded V2, in the presence of one equivalent of gC1qR, Trp fluorescence intensity still decreased upon addition of SR121463, but the decrease in width was significantly smaller. In fact, the shift between both curves (presence and absence of gC1qR) became wider as the antagonist concentration increased. This proportionality shows that the curve difference reflected a conformational rearrangement induced by antagonist binding. Adding i3_short dose-dependently displaced the profile of the fluorescence binding curves recorded in the presence of gC1qR towards the binding curves recorded in the absence thereof (Fig.

S3). The maximal effects are represented in Fig. 7b, at an SR121463 concentration of 10− 5 M.

Discussion Previous studies have shown that gC1qR is an interacting partner of the V2 and the β1-adrenergic receptors.36–38 In the case of the V2 receptor, this interaction is mediated by the i3 loop, and the arginine cluster within is supposed to be the main determinant.38 The V2 i3 loop thus appears as a critical determinant not only for the interaction with the GαS protein31 but also for the interaction with other cytoplasmic proteins. It is noteworthy that the isolated i3 loop peptide inhibits GαS signaling, in a competitive manner with regard to the whole receptor.20 In the absence of X-ray structure, examining receptor domains anchored to zwitterionic lipid micelles is a fruitful way to obtain structural features of GPCR cytoplasmic domains.22 Hence, we first solved the NMR solution structure of the V2 i3

NMR Structure of the GPCR V2 i3 Loop

500 Table 2 No. of experimental distance restraints Intraresidue Sequential (|i − j| = 1) Medium range (1 b |i − j| ≤ 4) Total NOE distance violations Number N0.3 Å Maximum violation (Å) Deviations from ideal covalent geometry Bond lengths (Å) Bond angles (°) Impropers (°) Average r.m.s.d. (Å) Backbone atoms (residues 241–246) Heavy atoms (residues 241–246) Backbone atoms (residues 248–255) Heavy atoms (residues 248–255) Ramachandran analysis (%) Most favored Additional allowed Generously allowed Disallowed

120 36 41 197 0 0.04 ± 0.01 0.0027 ± 0.0001 0.3873 ± 0.0116 0.1836 ± 0.0011 0.40 ± 0.18 1.77 ± 0.43 1.24 ± 0.66 2.30 ± 0.56 47.0 41.0 12.0 0.0

loop in DPC micelles to get insights into its molecular architecture. Secondly, we undertook a biophysical characterization of the complex between gC1qR and

i3_cyc and between gC1qR and a shorter peptide, i3_short, encompassing the arginine cluster. Our NMR structural study showed that in the presence of DPC micelles mimicking the plasma membrane, the i3 loop adopted a left-twisted α-helical hairpin conformation, where the N- and C-terminal parts formed well-defined packing helices extending about 17 amino acids each. Cyclic peptides have already been used in NMR studies to mimic the presumed fold of intracellular loops within entire receptors. Convergence of calculations is dependent on the number of structural constraints, and a lot of peptides corresponding to i3 loops, even cyclic, exhibit great conformational flexibility.19,22,28,52–55 Some other i3 loops adopt a well-defined structure 27,28,54,56–58 and i3_cyc apparently belongs to this second category. The original feature is its length (50 amino acids), because all other i3 intracellular loops whose isolated structure have been successfully solved are much shorter (to our knowledge, 24 amino acids). However, our work shows that length is not the only determinant of the flexibility of GPCR i3 loops. As N- and C-terminal helices were sufficiently long and well-defined to calculate a packing angle in between, we could bring to light that this left-twisted hairpin fold was present for the i3 loop in three crystal

Fig. 7. SR121463 antagonist binding to the entire purified V2 receptor monitored by Trp fluorescence spectroscopy. (a) Quenching of fluorescence as a function of SR121463 added concentration, recorded at 343 nM. Gray line, V2 receptor alone (c = 20 μM); dotted line, V2 receptor (c = 20 μM) in the presence of 1 equivalent of gC1qR. (b) Quenching of fluorescence (λ = 343 nM) of the V2 receptor (c = 20 μM) at an SR121463 concentration of 10− 5 M in the presence of gC1qR (c = 20 μM) and gC1qR (c = 20 μM) plus i3_short (c = 200 μM) and in the absence of response for unfolded V2R in 0.2% SDS. (c) Ribbon representation of the V2 receptor model. The side chains of Trp residues are represented as sticks or CPK spheres (for the most probable candidates of quenching upon binding to SR121463). The i3 loop is represented in yellow, and the side chains of the arginines of the binding site are represented and colored red.

NMR Structure of the GPCR V2 i3 Loop

structures: liganded bovine rhodopsin (1GZM), free bovine opsin (3CAP), and squid rhodopsin (1E12). The i3 loop of squid rhodopsin is particularly interesting to compare with, because it is of similar length (45 amino acids). It protrudes as a long rigid cytoplasmic extension of TM V and TM VI helices, forming a “rigid column” away from the membrane.14 This feature has been suggested to represent a molecular determinant to the coupling of squid rhodopsin with the Gαq subunit, as opposed to Gαt for bovine rhodopsin. Our data suggest that the coupling with the GαS protein may follow a similar mechanism. The main difference between the i3 loop structure solved in this study and the i3 loop of squid rhodopsin is the packing angle value, which was higher for the former. In fact, this larger value is ensured by a pronounced bending of the C-terminal helix in i3_cyc. At the membrane side, the packing angle is similar between both loops (squid rhodopsin = 30°, i3_cyc = 33°) and the divergence increases as one goes to the cytoplasmic side. We have constructed a structure of the V2 i3 loop based on the squid i3 loop template (data not shown), and we could indeed determine that the experimental interhelix NOE pattern was compatible with the helix orientation of this model at the membrane side but not at the cytoplasmic side. Hence, the difference in packing angle was not a cyclization artifact. The conservation of the packing angle sign of i3 loop enabled the grafting of the structure of the V2 loop onto the bovine rhodopsin structure, resulting in a structural model of the V2 receptor. Several peculiar features observed in the structure of the isolated i3 loop were more understandable in the whole receptor frame. The side chains of charged residues that were exposed to lipid chains in DPC micelles were now facing other TM helices (Gln225, Arg230, Glu231, Lys268, and Arg271), in contact with the polar head of the bilayer phospholipids (Arg230 and Lys268), or fully exposed to solvent (Glu242 and Glu258). Interestingly, mutation of most of the residues at the interface with other TMs (Gln225, Glu231, and Lys268) impairs coupling with the GαS protein (no report was found for Arg271).31,59 The arginine cluster Arg243-Arg252 itself belonged to the cytoplasmic interface of the V2 receptor, protruding high above the bilayer membrane and able to interact with gC1qR. Hence, the NMR structure of i3_cyc and the derived V2 model accounted for most biological experimental evidence we have been able to gather in the literature. Based on this structure, we can propose a new motif for GαS binding. Although the i3 loops of squid rhodopsin and V2 have similar length and do form both cytoplasmic helical “rigid columns”, the higher twist angle for V2 creates another different structural motif, with a larger binding cleft formed between the i1, i2, and i3 loops (Fig. 2d). In fact, insertion of residues in the central part of the V2 i3 loop diminishes the coupling activity with GαS.31 This decrease may be related to the destruction of the motif we present in this study. Altogether, our data confirm the biological relevance of the structural study of suitable fragments of GPCRs in the absence of crystals.60,61

501 We turned to various biophysical techniques to study the interaction between i3_cyc and gC1qR. Precipitation of either i3_cyc (in the presence of salt) or the complex hindered our investigation as high concentrations (10− 4 M). More specifically, stabilization of the complex required high salt concentrations in which the i3 peptide heavily precipitated. We were unable to conduct experiments following the complex formation without any precipitate formation, except when we labeled i3_cyc with the Alexa-488 fluorophore on its unique Lys residue (Lys248) and were thus able to work at submicromolar concentrations. Following the change in the fluorophore anisotropy induced by gC1qR binding allowed us to determine an interaction Kd of 1.8 ± 0.2 × 10− 6 M. We reasoned that the precipitation of the complex was due to the masking of the hydrophilic cluster of i3_cyc by gC1qR, whereas the hydrophobic helices were left in contact with water. We thus restricted our study to a shorter sequence of i3_cyc that contained the arginine cluster but was devoid of the most hydrophobic parts, i3_short. As expected, we no longer encountered any precipitation problem. Enzymatic proteolysis profiles of the gC1qR/i3_cyc and gC1qR/i3_short complexes showed identical profiles in delayed digestions. This similarity encouraged us to further investigate the gC1qR/i3_short complex. We were unable to label i3_short with a fluorophore, but given the relatively high Kd of the i3_cyc/gC1qR complex, we presumed that the Kd of the i3_short/ gC1qR complex had a similar value and undertook its NMR characterization by TR-NOE and STD spectroscopy. As expected, free and bound peptide resonances were found in fast exchange on the NMR chemical shift timescale. STD experiments allowed us to confirm that the arginine cluster represented the interaction surface with gC1qR. This conclusion was in agreement with previous biochemical data showing that the mutation of the arginines into alanines in the V2 receptor resulted in the loss of gC1qR binding.38 Interestingly, a recent study has shown that i3 loops of class A receptors are particularly rich in charged residues (Arg, Lys, and His).62 If gC1qR is a chaperone protein of GPCRs,41 such positively charged sequences could represent the binding site of misfolded receptors. Finally, TR-NOESY experiments showed that gC1qR binding promoted the folding of the otherwise flexible i3_short peptide into a helix-like conformation. Other TR-NOE studies have investigated the interaction between GPCR intracellular fragments with target proteins such as the GαI52 or β-arrestin proteins 63,64 and showed the folding of a disordered peptide upon binding. In fact, a large portion of the cytoplasmic region of GPCRs is predicted to be disordered in silico, but the residue composition is different from other intrinsically disordered proteins.62 This unusual amino acid distribution may be related to the capacity of those more or less flexible segments to become well structured when bound to their targets. Because the corresponding segment to i3_short was more flexible than the rest of the structure in i3_cyc,

502 we introduced the TR-NOE observed in the gC1qR/ i3_short complex in the constraint list of i3_cyc. Calculations suggested that, as expected, gC1qR binding would rigidify the linker connecting the hairpin helices. They also suggested that the structure observed for the gC1qR-bound state of i3_short was not compatible with the N- and C-terminal helix orientation of i3_cyc. Of course, it is possible that the reorientation could be strictly local to the furthest cytoplasmic side of the hairpin helices. However, more and more pieces of evidence are presented for GPCRs, which suggest that TM and cytoplasmic helices behave as semirigid bodies (see the conformational differences between liganded and free opsin, for example).9 Moreover, we have performed additional fluorescence experiments, which favor the hypothesis of a more global remodeling of V2 helices. Binding of SR121463to the whole V2 receptor was accompanied by a diminution of the tryptophan fluorescence. Mutation data and modeling studies indicate that the binding site for SR121463 is a hydrophobic pocket between TM III, TM IV, TM V, and TM VI.65,66 In the models, Trp284 (TM VI) interacts directly with the antagonist and, hence, is an evident candidate for the fluorescence quenching we observed in the present study (Fig. 7c). Other Trp residues (e.g., Trp193-EL2, as well as Trp208-TM V, Trp293-TM VI, or Trp296-EL3) may also see their environment change through an antagonist-driven conformational remodeling of the receptor. Interestingly, the presence of gC1qR in the assay led to a decrease in the amplitude of the fluorescence quenching. As evidenced on Fig. 7c, the i3 loop of V2 contains no Trp residue. Two are located in the i1 and i2 loops (C-terminal part); the rest belongs to TM helices or extracellular loops. All of them are located at a minimal distance of 32 Ǻ from the arginine cluster, and it is unlikely that one of them directly interacts with gC1qR. Hence, two phenomena could potentially account for the shift induced by gC1qR in the fluorescence assay: (i) upon antagonist binding, a remodeling of the V2 cytoplasmic binding interface to gC1qR occurred, provoking, in turn, a change in the fluorescence emission of Trp residues of gC1qR; (ii) upon gC1qR binding, there was a long-distance conformational rearrangement of V2, such that the environment of the Trp residues of V2 changed, and hence their emission response to antagonist binding. It is of note that if hypothesis (i) is true, it also indirectly confirms that the structure of the gC1qRbound hairpin linker is compatible only with a limited range of TM helix orientations, as antagonist binding would then cause its remodeling. Three experimental pieces of evidence, however, favor (ii) over (i). Hypothesis (ii) is consistent with the fact that gC1qR addition did not affect the barycenter of the fluorescence binding curve, as might be expected in the case of a burial of gC1qR Trp residues in contact with V2. Hypothesis (ii) is also in keeping with the fact that gC1qR binding by V2 is ligand independent. Finally, the retrieval of the original level of fluorescence quenching in the presence of i3_short also favors the hypothesis where the emission differences originate

NMR Structure of the GPCR V2 i3 Loop

from V2 Trp residues. Hence, our data suggest that a small structural change at the intracellular side (here, a different folding of the hairpin linker) induced by the binding of a cytoplasmic protein (here, gC1qR) can be efficiently propagated towards the TM domains by a reorientation of TM helices.

Materials and Methods Peptide preparation and purification The i3_cyc peptide encompassing the Gln225-Leu274 sequence of the V2 rat receptor and the i3_endo peptide corresponding to the sequence Thr279-Lys306 of the rat ETA endothelin receptor third intracellular loop were synthesized by Fmoc solid-phase peptide synthesis as previously described.20,32 Briefly, a bromoacetic acid was introduced to the N-terminus of the Gln225-Thr274-Cys-Gly peptide. Cyclization was then achieved by a nucleophilic substitution of the Br atom by the thiol group. All methionines were replaced with norleucines. The i3_short peptide corresponding to the Ser241-Ser254 sequence of the V2 receptor, that is, the central portion of i3_cyc, was purchased from EZB Biolab, Inc. Sequences of i3_cyc and i3_short are depicted in Fig. 1a. Fluorophore labeling An excess of Alexa-488 C5-succinimidyl ester (2.7 equivalents) was covalently coupled to the unique lysine residue of i3_cyc, namely, Lys268, in a 1-h reaction in DMSO at pH 8.3 and room temperature. After acidification with trifluoroacetic acid, the labeled peptide was separated from excess dye and unlabeled peptide by HPLC chromatography and its concentration was determined by UV spectroscopy. gC1qR protein purification Recombinant gC1qR protein (amino acids 74–282, with Leu68 substituted by a Met residue) was expressed in Escherichia coli BL21 from plasmid pT7 (from Adrian R. Krainer of the Cold Spring Harbor Laboratory, New York). Purification was done following Krainer's laboratory protocol.35 V2 purification and refolding The His-tagged h-V2 receptor production has been patented [Mouillac B., Sen T. & Banères J. L. (29 Dec 2004). Method for producing a recombinant protein of interest and protein thus produced. In INSERM/CNRS (ed). N°WO2004/113539 A3, France]. The corresponding article is in preparation. Limited proteolysis experiments gC1qR (20 μM) in 10 mM sodium phosphate buffer, pH 7.5, was incubated with various amounts of i3_cyc, i3_short, and i3_endo peptides in the presence of 0.40 μM chymotrypsin for 10 min at room temperature in 200-μl aliquots. Digestion was stopped by the addition of 1 M Tris buffer at pH 7. Fractions were resolved by SDS gel electrophoresis and visualized by Coomassie blue staining.

NMR Structure of the GPCR V2 i3 Loop Trp fluorescence experiments Tryptophan fluorescence measurements were performed on a Safire II (Tecan Instruments). Spectra were recorded at 25 °C. Emission spectra were obtained by excitation at 295 nm, with emission monochromator scanning between 320 and 380 nm with an increment of 1 nm and an integration time of 0.5 s. Buffer composition was as follows: 20 mM Tris, pH 8.8, 150 mM NaCl, 0.058% Foscholine C12, 0.01% dodecylmaltoside, and 0.01% cholesterolhemisuccinate. The concentration of V2 was 20 μM. For the V2–gC1qR complex, gC1qR concentration was 20 μM. The fluorescence contributions from gC1qR and buffer components were subtracted when needed. Titration with the SR121463 antagonist was performed by adding small aliquots from a solution stock in DMSO (c = 1 mM) up to a final concentration of 60 μM. Fluorescence anisotropy experiments Fluorescence intensity and anisotropy measurements were carried out on a Safire II (Tecan Instruments) equipped with dual monochromators and the ability to rapidly collect data from 60-μl samples in 96-well flat-bottom black microplates. For the determination of the dissociation constant (Kd) of the i3_cyc/gC1qR complex, samples of Alexa-488–i3_cyc in complex with gC1qR were excited at 470 nm and emission was measured at 530 nm. Buffer composition was 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 5 mM dithiothreitol, 0.5% Triton X-100, and 10% (v/v) glycerol. The measurements were initiated at the highest concentration of gC1qR protein and then the sample was diluted successively by a factor of 0.75 with a solution containing 2 nM fluorescent peptide. The reported values are the average of two measurements with two different protein samples. Binding data were analyzed using the package BIOEQS assuming a 1:1 stoichiometry.68

503 of a TOCSY NH/Hβ correlation peak in the presence and absence of Gd(DTPA-BMA), respectively. Ai = 1 if the residue is not exposed (maximal value) and tends towards 0 as the solvent exposure increases.73 1D STD NMR spectra50 were recorded on a mixture of i3_short and gC1qR with 2048 scans and selective saturation of gC1qR resonances at 6.7 ppm (−30 ppm for reference spectra). An irradiation test was also performed on a free peptide sample to verify that only gC1qR resonances were irradiated. The i3_short/gC1qR ratio was increased from 1:80 to 1:16. Investigation of the time dependence of the saturation transfer from 0.5 to 4 s with equally spaced 50-ms Gaussian shaped pulses (separated by a 1-ms delay) showed that 2 s was needed for efficient transfer of saturation from gC1qR to the i3_short peptide. As no baseline distortion was observed, no T1ρ filter was applied to eliminate background resonances of gC1qR. Subtraction of free induction decay values with on- and off-resonance protein saturation was achieved by phase cycling. A relaxation delay of 1.41 s (Aq + D1) and 128 dummy scans were employed to reduce subtraction artifacts. STD amplification factors, Si, were introduced:49,50 Si ¼ ðI0  Isat Þ=I0  ligand excess where I0 and Isat are the intensities of one signal in the reference NMR spectrum and in the on-resonance spectrum, respectively. TR-NOESY spectra67 of the i3_short/gC1qR complex were recorded with 4K points and 1K t1 increments and a relaxation delay of 1.1 s. The optimal conditions for the TRNOESY measurements were determined by considering a peptide/protein ratio ranging from 5 to 100 with mixing times (τm) of 100, 200, and 300 ms. The final NMR spectra were acquired with a peptide/protein ratio of 80 (ci3_short = 4 mM, cgC1qR = 50 μM) and a mixing time of 300 ms. All postprocessing was performed with the Gifa software,74 and visualization was done with the NMRView package.75

NMR spectroscopy

Structure calculations

Proton NMR experiments for the assignment of the resonances of i3_cyc (50 amino acids) were recorded on an Innova 800-MHz spectrometer at 35 and 45 °C. Other NMR experiments were carried out on a Bruker 600-MHz Avance spectrometer equipped with a z-gradient 13C–15N–1H tripleresonance cryoprobe at 20 °C. 2D total correlated spectroscopy (TOCSY)69 (mixing time, 35–60 ms), NOESY70,71 (mixing time, 100 and 200 ms for i3_cyc, 300 ms for i3_short), 13 C–1H heteronuclear single quantum coherence (HSQC), and TOCSY–HSQC were recorded to obtain the resonance assignment. Proton and carbon chemical shifts were referenced relative to 2,2-dimethyl-2-silapentane-5-sulfonic acid resonances.72 For i3_cyc, the peptide concentration was 1 mM in the presence of 100 equivalents of deuterated DPC (Eurisotop, France) at pH 4 (pD 4) in 90% H2O:D2O (100% D2O). A control experiment 13C–1H HSQC–TOCSY with 200 equivalents of DPC was also performed in order to verify that in this concentration range, there was no chemical shift dependence on DPC concentration (Fig. S5). Slowly exchanging amide protons were identified by recording 50-ms TOCSY spectra at different delays after addition of D2O to the lyophilized sample. Identification of solvent-exposed residues was obtained by recording a TOCSY spectrum of i3_cyc in the presence of 100 equivalents of DPC and 5 mM Gd(DTPA-BMA) (trade name, Omniscan from Nycomed).45 An attenuation factor Ai was calculated for each residue i with Ai = Vip/Vid, where Vip and Vid stand for the volume

All structure calculations were performed with the Aria 2.0 software76,77 interfaced with the CNS program.78 The topologies of the modified amino acids (norleucine, D-glutamine) and of the thio-ether bridge between the C-terminal cysteine residue and the N-terminal glycine were constructed based on comparison with leucine, glutamine, and cysteine residues and with available databases. The cross peaks in the NOESY spectra were pick-peaked while comparing the presence and intensity of signals at various mixing times to avoid peaks due to magnetization transfer by spin diffusion. The NOE cross peaks were volume integrated by using the routine in NMRView. In addition, 40 dihedral angle restraints were obtained by using the TALOS program79 with HN, Hα, 13Cα, and 13Cβ chemical shifts as input, after verification that inclusion of chemical-shiftderived constraints did not alter the overall fold but only accelerated the convergence, quality, and precision of structure calculations.80 The constraints were introduced for structure calculation with the standard protocol of Aria 2.0, with 100 structures calculated per iteration, of which the 20 best structures were selected for further evaluation. The final refinement stage in H2O was bypassed for i3_cyc calculations as we considered it not being appropriate for a micelle-bound structure. Because the NMR spectra of i3_cyc were crowded, numerous calculations were launched. In the initial calculations, the constraint list contained approximately 10% of unassigned NOEs (for a total of 826).

NMR Structure of the GPCR V2 i3 Loop

504 Proposal of assignment by Aria was then manually checked and validated. For DPC-bound i3_cyc, the final list of NOE constraints contained 469 intraresidue, 199 sequential, 141 medium-range, and 17 long-range constraints and 8 ambiguous NOEs. Based on the visual inspection of the initial calculated structures and on the identification of slowly exchanging amide protons, hydrogen-bond upperdistance limitations for 22 residues between the O(n) and H (n + 3) and O(n) and N(n + 3) of 1.8 and 2.8 Å, respectively, were added to the list of restraints for the final structure calculations. For gC1qR-bound i3_short, the final list contained 197 unambiguous NOE constraints (129 intraresidue, 36 sequential, and 41 medium range). The constraints obtained for the i3_short peptide when bound to gC1qR were also introduced for calculations of i3_cyc, after suppression of the original constraints for the relevant segment (Ser241-Ser255), in order to get insight into the conformational changes induced by gC1qR binding for the whole third intracellular loop. Calculations were launched with and without the 18 experimental NOEs between the segment and the hairpin helices. Recalculations of isolated i3_cyc structures were also performed, omitting these 18 NOEs and the TALOS-derived constraints. Isolated i3_cyc and gC1qR-bound i3_cyc structures were, however, insensitive to these constraints. In a subsequent step, RDCs (corresponding to an alignment tensor of 20 Hz for its axial component) were also added to the constraint list of gC1qRbound i3_cyc, to freeze the N- and C-terminal helices in their original orientation as in isolated i3_cyc. At this stage, residues Ser241 and Glu255, which are the terminal residues of i3_short, were left free (or not) of any constraint to let a chance for the substructure of gC1qR-bound i3_short to get accommodated in the i3_cyc hairpin structure. The quality of the final structures was assessed with the program PROCHECK.81 Molecular modeling A model of the V2 receptor was derived from the NMR solution of DPC-bound i3_cyc (this study) and the crystallographic structure of the bovine rhodopsin5 [Protein Data Bank (PDB) code: 1GZM)] introduced as templates for the Modeller software.82 V2 TMs were predicted by the TMHMM version 2.0 software.83 Alignment of the TM domains of the V2 receptor onto the TM domains of bovine rhodopsin was done on the basis of the most conserved residues/sequences motifs across the Class A GPCRs.84,85 The C-terminal segment T269VRMT273 of the DPC-bound structure of i3_cyc was spatially superimposed onto the V249TRMV253 segment of the opsin structure. Visual inspection revealed that the V2 Q225VLI228 segment (corresponding to the N-terminal extremity of i3_cyc structure) had to be superimposed onto the opsin Y223GQL226 segment to achieve optimal grafting of the NMR-derived intracellular loop on the crystal template. The i2 loop of the V2 model was modeled with the opsin i2 loop as template based on the alignment of the DRH/ERY motif in the N-terminal extremity. Another model was constructed using the structure of the β2-adrenoreceptor as template (PDB code: 2RH1) and was very close to the rhodopsin-derived model. PDB accession number NMR solution structures and structural restraints have been deposited into the Research Collaboratory for Structural Bioinformatics PDB under accession number 2JX4.

Acknowledgements G.B. and S.G. were recipients of a Ministère de l'Education Nationale de la Recherche et de la Technologie grant from the French government. We want to thank the mass spectrometry platform of the “Institut de Génomique Fonctionelle” for their aid.

Supplementary Data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/ j.jmb.2009.02.065

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