The Conformational Equilibrium of the Neuropeptide Y2 Receptor in Bilayer Membranes

Abstract Dynamic structural transitions within the seven‐transmembrane bundle represent the mechanism by which G‐protein‐coupled receptors convert an extracellular chemical signal into an intracellular biological function. Here, the conformational dynamics of the neuropeptide Y receptor type 2 (Y2R) during activation was investigated. The apo, full agonist‐, and arrestin‐bound states of Y2R were prepared by cell‐free expression, functional refolding, and reconstitution into lipid membranes. To study conformational transitions between these states, all six tryptophans of Y2R were 13C‐labeled. NMR‐signal assignment was achieved by dynamic‐nuclear‐polarization enhancement and the individual functional states of the receptor were characterized by monitoring 13C NMR chemical shifts. Activation of Y2R is mediated by molecular switches involving the toggle switch residue Trp2816.48 of the highly conserved SWLP motif and Trp3277.55 adjacent to the NPxxY motif. Furthermore, a conformationally preserved “cysteine lock”‐Trp11623.50 was identified.

Preparation of E. coli cell extract for CF expression. The S12 extract used in CF expression was prepared from E. coli Rosetta(DE3). Cells were cultivated in 25 ml of 2×YTPG medium with 34 µg/ml chloramphenicol. 2×YTPG medium consisted of 16 g/l tryptone, 10 g/l yeast extract, 5 g/l NaCl, 22 mM NaH2PO4·2 H2O, 40 mM Na2HPO4, and 1 % (w/v) glucose. The cells were transferred in a 200 ml overnight culture, then in a 200 ml fermentation starter culture, and finally at an OD600 of 1.5 with a dilution of 1:20 into the fermenter (with a total volume of cell medium of 3 l). Cells were cultivated at 37°C, at pH 7, with an air flow of 8 l/min, and a stirring rate of 600 to 800 rpm. Cells were harvested on ice after they had reached an OD600 of ~3.5 to 4.0. They were centrifuged at 5,000 g and 4°C for 15 min. The pellet was resuspended twice in S30 extract buffer (10 mM Tris-acetate, 14 mM magnesium acetate, 60 mM potassium acetate, pH 8.2) followed by centrifugation at 4,000 g, and 4°C for 15 min. Finally, the pellet was resuspended in 10 ml of S30 extract buffer per 8 g of cells and the suspension stored in Falcon tubes on ice overnight in the coldroom. Before cell lysis, 1× cOmplete TM (EDTA-free Protease Inhibitor Cocktail) and 1 mM DTT were added to the solution. Cell lysis was accomplished by an APV 2000 homogenizer. After centrifugation at 12,000 g and 4°C for 30 min, the supernatant was incubated at 30°C and 150 rpm for 2 h. The supernatant was placed into a dialysis bag (MWCO 12-14 kDa, Zellu Trans Roth) and dialyzed against a 40-fold excess of S30 extract buffer supplemented with 1 ml/l 2-Mercaptoethanol in two steps at 4°C. The extract was snap frozen in liquid nitrogen in aliquots of 800 µl and stored at -80°C until usage.
Purification and reconstitution of Y2R. After 24 h of CF expression, precipitated Y2R was solubilized by addition of 9 ml 50 mM sodium phosphate buffer (pH 6.5), 15 mM SDS, and 50 mM DTT per 1 ml reaction mix. The excess of DTT was removed in a two-step dialysis against the same buffer without DTT. The protein solution was adjusted to pH 8 by NaOH and the denatured protein purified by immobilized metal affinity chromatography using a 5 ml HisTrap TM HP column (GE Healthcare, Germany). [4] The buffers used in chromatography consisted of 50 mM sodium phosphate and 15 mM SDS, with pH 8 for the equilibration buffer and pH 4 for the elution buffer. Protein yields of 0.7 to 1.4 mg per 1 ml reaction volume were determined by UV absorption (NanoDrop, Spectrophotometer ND-1000, peqlab Biotechnologie GmbH). Protein purity was verified by SDS PAGE analysis. Y2R was reconstituted in two steps as described: [4] first, the SDS concentration was reduced by dialysis and the disulfide bond was formed by addition of a glutathione redox system (0.2 mM reduced glutathione, 0.1 mM oxidized glutathione) to a carefully degassed buffer containing 50 mM sodium phosphate, 2 mM SDS, 1 mM EDTA (pH 8.5). Subsequently, the receptor was concentrated by addition of 25 % (w/v) PEG20,000 to the same buffer. Second, the receptor was incorporated into preformed lipid bicelles of 1,2-dimyristol-sn-glycerol-3-phosphocholine (DMPC or chain-deuterated DMPC-d54) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC) in 50 mM sodium phosphate (pH 7) by fast temperature changes from 42°C to 0°C. The molar ratio was 1:200:800 of receptor:DMPC:DHPC. Lipids were purchased from Avanti Polar Lipids via Merck (Darmstadt, Germany). After removal of residual detergent using BioBeads SM2 (Bio-Rad, München/Germany), the samples were centrifuged at 21,500 × g. [4] For experiments in the presence of NPY, Y2R reconstituted in lipid bicelles was incubated with the peptide ligand in a molar ratio of 1:1.5 overnight at room temperature. Finally, the samples were centrifuged and the pellets loaded into the MAS rotors.
Peptide synthesis. NPY was synthesized by automated solid phase peptide synthesis following the Fmoc/tert-butyl strategy as described before. [5] In brief, the peptide sequence is built up from C-to Nterminus in 15 μmol scale on Rink amide resin by repetitive cycles of Nα-Fmoc deprotection, activation, and coupling of the next amino acid in a Syro II peptide synthesizer (MultiSynTech, Witten/Germany). All reactive side chains carried protection groups that were stable under the conditions of the Fmoc cleavage. Fmoc was cleaved by successive treatment with 40% piperidine and 20% piperidine in N,Ndimethylformamide (DMF) for 3 and 10 minutes, respectively. The amino acids were conjugated in a double coupling procedure (2 x 45 min) with 8 eq. Nα-Fmoc-amino acid in situ activated with 8 eq. ethyl 2-cyano-2-(hydroxyimino)acetate and 8 eq. diisopropylcarbodiimide in DMF.
The peptides were cleaved off the resins by using trifluoroacetic acid/H2O/triisopropyl silane (90/5/5, v/v/v) for 2 hours, which simultaneously removes all remaining side chain protection groups. The peptides were precipitated using ice-cold diethyl ether. The identity of the peptides was confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry (Ultraflex III MALDI-TOF/TOF; Bruker, Billerica/USA), and the peptides were purified to 95% by reversed-phase high performance liquid chromatography as reported previously. [5] Functional characterization of Y2R. A fluorescence polarization assay was carried out to determine the ligand binding capacity of reconstituted Y2R obtained from CF expression. [4,7,8] The receptor was reconstituted in DMPC/DHPC bicelles at a molar ratio of 1:600:2400 in 50 mM sodium phosphate buffer pH 7. Subsequently, various concentrations of Y2R were incubated with 50 nM TAMRA-[Ahx5-24]NPY overnight. This peptide variant is a reduced-size Y2R-specific agonist, which activates Y2R-expressing HEK293 with nanomolar potency (pEC50 8.87 ± 0.17 vs 10.35 ± 0.08 of NPY). Measurements were carried out on a FluoroMax-2 (JOBIN YVON) spectrometer in a 10 mm quartz cuvette at 20°C with linear polarized light, an excitation wavelength of 549 nm, an emission wavelength range of 574 to 578 nm, and 90° detection angle. Three independent measurements were carried out in triplicate. A sigmoidal dose-response curve was used to fit the data with the Origin software.
Preparation of Y2R + NPY + arrestin-3 sample. For NMR experiments of Y2R in the presence of arrestin, the phosphorylation-independent variant of bos taurus arrestin-3 [9] was added. This variant contained the 3A mutation (Ile397Ala, Val398Ala, Phe399Ala) as described previously. [9] The modified arrestin-3 was prepared as described. [10] Briefly, the protein was expressed in E. coli Rosetta(DE3) or E. coli NiCo21(DE3) cells in LB medium at 26°C and 150 rpm. Expression was induced by addition of IPTG to a final concentration of 35 µM at an OD600 of ~1.0 to 1.5. A multistep cell lysis included the addition of lysozyme (Roth, Karlsruhe/Germany), freezing at -80°C, sonication, incubation with 8 mM MgCl2 plus DNase (Sigma, Taufkirchen/Germany) and several centrifugation steps. The protein was precipitated by the addition of ammonium sulfate to a final concentration of 2.4 M, pelleted, and dissolved in column buffer. The following chromatography steps included purification on a heparin-Sepharose column, Q-and SP-Sepharose columns (GE Healthcare). The purification steps were validated by SDS-PAGE and Western blot.
For incubation with Y2R, arrestin-3 was dialyzed in two steps against 50 mM sodium phosphate (pH 7), 1 mM EDTA, and 200 mM NaCl. The ternary complex was formed by incubation of Y2R reconstituted in DMPC/DHPC bicelles with NPY (twofold molar excess) and arrestin-3 (threefold molar excess) overnight at 4°C. The solution was centrifuged and the pellet loaded into the MAS rotor.
Binding of arrestin-3 to Y2R was verified by a pull-down assay. To this end, the proteins were separated from the lipids by chloroform/methanol precipitation and the pellet resolved in SDS sample buffer. The samples were analyzed via SDS-PAGE and Western Blot. The F431 rabbit polyclonal antibody [11] was used for detection of arrestin-3.
NMR spectroscopy. Membrane samples containing up to 6 mg of isotopically labeled and reconstituted Y2R were subjected to NMR spectroscopy. The samples were filled into 3.2 mm MAS rotors. Bruker Avance III 600 MHz and Avance Neo 700 MHz NMR spectrometer using a triple resonance MAS probes with a 3.2 mm spinning module were used. Homonuclear 13 C-13 C DARR correlation experiments [12] were performed at a temperature of -30°C and MAS frequencies of 11 or 12 kHz with a mixing time of 10 ms and a CP contact time of 2 ms. The DipShift pulse sequence [13] was applied at an MAS frequency of 5 kHz with the 90° pulses adjusted to 4 µs for both channels. CP contact times of 200 µs, 700 µs or 2 ms were used for the excitation of the 13 C nuclei. For decoupling, the spinal sequence (heteronuclear 1 H-13 C decoupling [14] ) and the FSLG sequence (homonuclear 1 H-1 H decoupling [15] ) at an radiofrequency field strength of ~80 kHz were applied. To ensure objective data evaluation, spectral intensity was integrated over defined spectral regions based on the peaks of the 13 C-13 C DARR NMR spectra. The order parameters from the DipShift experiments resulted from the ratio of the motionally averaged dipolar coupling strength and the rigid limit of the dipolar coupling. [16] The dipolar coupling strength was derived from best fit of numerically simulated dipolar dephasing curves fitted to the experimental dephasing curves measured over one rotor period (9 increments). At least two independent samples were prepared for each conformational state of Y2R. The molecular order parameters were converted into amplitude information using the formula S = cos(θ) (1 + cos(θ))/2. [17] DNP experiments. Samples of Y2R labeled with 13 C-Trp and either 15 N-Lys, 15 N-Gly, 15 N-Leu, 15 N-Pro, 15 N-Ser, or 15 N-Met and reconstituted into DMPC-d54 membranes were pelleted via centrifugation and shipped on dry ice to Frankfurt/Main (Germany) within two days. The wet pellets were incubated with 50-100 µl of 20 mM AmuPOL in Glycerol-d8:D2O:H2O (30:60:10) at 4°C overnight. [18] The supernatant was removed and the soaked pellet was packed into 3.2 mm ZrO2-MAS rotors. DNP experiments were recorded using a Bruker 400 MHz DNP system consisting of a 400 MHz WB Avance II NMR spectrometer and a 263 GHz Gyrotron as a microwave source equipped with a 3.2 mm HCN low temperature MAS probe. All experiments were recorded at an MAS frequency of 8 kHz at a temperature of around -164°C. The microwave power at the probe was 10 W. Spectra were referenced indirectly to DSS using the right signal of the glycerol-d8 at 64.78 ppm. All experiments were recorded with 100 kHz high power decoupling, CP contact times of 1 ms, a recycle delay of 1.2-1.8 s (depending in the 1 H T1 times of the samples), a 4 ms specific NCO transfer and 20 ms PDSD mixing for the CX step. Typically 40,000 and 180,000 scans were recorded for each sample. Figure S1. Summary of analytical and pharmacological data on protein preparation of Y2R by cell-free expression and formation of the ternary complex of Y2R+NPY+arrestin-3. Precipitated Y2R obtained by cellfree expression was solubilized in SDS, purified by immobilized metal affinity chromatography and eluted by a pH shift. Panel A) reproduces a typical chromatogram of the purification B) SDS-PAGE of purified Y2R (Y2R shows a prominent band with a molecular weight of about 44 kDa). C) Fluorescence polarization assay to monitor ligand binding to the reconstituted Y2R. [6] Varying Y2R concentrations were incubated with 50 nM of TAMRA-labeled [Ahx5-24]NPY. Only weak ligand binding to empty lipid bicelles (in the absence of Y2R) is observed. The Y2 receptor binds [Ahx5-24]NPY with similar nanomolar affinity (Ki = 13 nM) in cellular membranes. [19] D) SDS-PAGE showing the ternary complex of Y2R+NPY+arrestin-3 as visualized after a pulldown assay. E) Western Blot analysis to monitor the presence of arrestin-3 in the sample Y2R+NPY+arrestin-3. Arrestin-3 was detected with the F431 rabbit polyclonal antibody. [10] The control shows purified protein.    Figure 1 and Figure 2): Preparation sheet for cell-free expression of 13 C/ 15 N-Trp-labeled Y2R. Compounds were mixed in a master mix split into a feeding and a reaction mix. The feeding mix and the reaction mix are supplemented with further reactants to a final volume of 17 ml and 1 ml, respectively.