Functional reconstitution of the HIV receptors CCR5 and CD4 in liposomes Franc¸ois Devesa, Vida Chams, Premkumar Dinadayala, Alexandre Stella, Aude Ragas, Henri Auboiroux, Toon Stegmann and Yannick Poquet Institut de Pharmacologie et de Biologie Structurale; CNRS UMR 5089, Toulouse, France Reconstitution of membrane proteins allows their study in a membrane environment that can be manipulated at will. Because membrane proteins have diverse biophysical pro- perties, reconstitution methods have so far been developed for individual proteins on an ad hoc basis. We developed a postinsertion reconstitution method for CCR5, a G protein coupled receptor, with seven transmembrane a helices and small ecto- and endodomains. A His 6 -tagged version of CCR5 was expressed in mammalian cells, purified using the detergent N-dodecyl-b- D -maltoside (DDM) and reconstitu- ted into preformed liposomal membranes saturated with DDM, removing the detergent with hydrophobic polysty- rene beads. We then attempted to incorporate CD4, a protein with a single transmembrane helix and a large hydrophilic ectodomain into liposomal membranes, together with CCR5. Surprisingly, reconstitution of this protein was also achieved by the method. Both proteins were found to be present together in individual liposomes. The reconstituted CCR5 was recognized by several monoclonal antibodies, recognized its natural ligand, and CD4 bound a soluble form of gp120, a subunit of the HIV fusion protein that uses CD4 as a receptor. Moreover, cells expressing the entire fusion protein of HIV bound to the liposomes, indicating that the proteins were intact and that most of them were oriented right side out. Thus, functional coreconstitution of two widely different proteins can be achieved by this method, suggesting that it might be useful for other proteins. Keywords: membrane protein; reconstitution; liposome. Reconstitution of membrane proteins allows the study of their behaviour in a membrane in the absence of other proteins, and the manipulation of their concentration and environment. Unfortunately, no single reconstitution method is applicable to all membrane proteins, most likely because their biophysical character varies. The most frequently used method is coinsertion reconstitution, in which detergent is removed from a mixture of detergent- solubilized protein and lipid [1,2], a method which has led to the successful reconstitution of membrane proteins with diverse structures [3–6]. However, no common conditions are so far known that would allow a standard operating procedure for reconstitution by this method to be esta- blished. Postinsertion reconstitution is a fundamentally different method based on the removal of detergent from detergent-solubilized protein added to a preformed lipo- some incubated with a detergent at a concentration almost leading to the onset of membrane solubilization [1]. Although the physical basis for this type of reconstitution is not clear, a number of proteins have now been reconsti- tuted by this method [2,7–14]. The abundant proteins belonging to the superfamily of G-protein coupled receptors (GPCR) that possess seven transmembrane ahelices, have important roles in eukary- otic signalling. Few GPCR have been reconstituted, by a variety of coinsertion or postinsertion protocols, with variable results [4,15–17]. The b-chemokines RANTES, MIP-1a,andMIP-1b are recognized by the GPCR CCR5 [18–21]. Through binding to the chemokine, this receptor plays a crucial role in inflammatory processes. CCR5 is also used as a receptor by primary strains of HIV-1 [21–24]. When infecting a host cell, the membrane protein complex of HIV-1, gp120/gp41, first binds to CD4 [25,26], an integral membrane protein with a single transmembrane a-helix. Binding induces a conformational change in gp120/ gp41 [27,28], which leads to an increased affinity for CCR5 present in the same host cell membrane [29–31]. Further conformational changes induced by the latter interaction then induce fusion between the viral and the cellular membrane, allowing the virus to enter its host cell [32]. In many cell membranes, CCR5 and CD4 are associated, and this association may be important to HIV infection [33]. In this paper, we describe a postinsertion reconstitution method that allows simultaneous incorporation of both CCR5 and CD4, isolated from a mammalian cell in which they were expressed together, into a single proteoliposomal membrane. Proteoliposomal membranes containing both proteins recognized the natural ligands for CCR5, a conformation-specific antibody recognized CCR5, the recon- stituted CD4 bound HIV gp120, and the proteoliposomes Correspondence to Y. Poquet, Institut de Pharmacologie et de Biologie Structurale; CNRS UMR 5089, 205 Route de Narbonne, 31077 Toulouse Cedex, France. Fax: + 33 5 61 17 59 94, Tel.: + 33 5 61 17 54 64, E-mail: yannick.poquet@ipbs.fr Abbreviations: Cmc, critical micelle concentration; DDM, N-dodecyl- b- D -maltopyranoside; Egg-PtdEth, egg phosphatidylethanolamine; Egg-PtdCho, egg phosphatidylcholine; N-NBD-PtdEth, N-(7-nitro- 2,3,1-benzoxadiazol-4-yl)-phosphatidylethanolamine; N-Rh-PtdEth, N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine; SM, egg sphingomyelin; GPCR, G-protein coupled receptor; DMEM, Dulbecco’s modified Eagle’s MEM. (Received 14 June 2002, revised 13 August 2002, accepted 29 August 2002) Eur. J. Biochem. 269, 5163–5174 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03213.x bound strongly to cells expressing the HIV fusion protein. Therefore, functional reconstitution of different types of membrane proteins was achieved by our method. MATERIALS AND METHODS Materials Egg phosphatidylcholine (egg-PtdCho), egg phosphati- dylethanolamine (egg-PtdEth), egg-sphingomyelin (SM), cholesterol, N-(lissamine rhodamine B sulfonyl)-phosphati- dylethanolamine (N-Rh-PtdEth), and N-(7-nitro-2,3,1-ben- zoxadiazol-4-yl)-phosphatidylethanolamine (N-NBD-PtdEth) were purchased from Avanti Polar Lipids (Birmingham, Ala.). Detergents were obtained from Sigma, the chemo- kines MIP-1a and RANTES from R & D Systems (Wiesbaden, Germany), and 125 I-labelled MIP-1a from NEN Life Science Products (Paris, France). Anti-myc mAb 9E10, fluorescein isothiocyanate (FITC) conjugated goat anti-mouse Ig, anti-CD4 Q4120 and the anti-His 6 Ig His1 were purchased from Sigma, horse-radish peroxidase con- jugated goat anti-mouse Ig from Biorad (Marnes-la-Co- quette, France), 125 I-labelled sheep anti-mouse Ig from Amersham Pharmacia Biotech (Saclay, France), anti-CCR5 mAb 2D7 from PharMingen Becton-Dickinson (Le Pont des Claix, France), and anti-CCR5 mAbs 181 (Mab181) and 182 (Mab182) from R & D Systems. Paramagnetic beads coupled to anti-mouse Ig or anti-CD4 Igs were purchased from Dynal (Compie ` gne, France). NIH 3T3.T4.CCR5 cells and cells expressing a soluble version of the gp120/gp41 of strain JR-FL (CHO JR-FL gp160, clone A19) were obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH through D. R. Littman and J. Arthos, respectively. HeLa cells expressing the gp120/gp41 from strain ADA was the kind gift of E. Bahraoui, Universite ´ Paul Sabatier, Toulouse, France. Construction and stable expression of HIV receptor vectors Plasmid DNA containing the CCR5 gene was obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH from N. Landau (ref no. 3325). For the construction of a tagged version of CCR5, an XhoI restriction site was introduced upstream of the start codon of CCR5 and an ApaI site upstream of the stop codon by PCR and expanded in pBSK1 plasmid. The resulting XhoI/ApaI fragment was cloned into the corres- ponding sites in the polylinker region of the pcDNA3.1- myc-His expression vector (Invitrogen, Groningen, the Netherlands) in order to obtain a recombinant protein containing the C-myc epitope followed by six histidines at the C-terminus. A plasmid containing the wild type version of CCR5 was constructed by introducing the coding sequence of CCR5 between the ApaIandHindIII restric- tion sites of a pRC-CMV vector (Invitrogen). For protein expression, CHO cells grown in Dulbecco’s modified Eagle’s Medium (DMEM), containing 10% foetal calf serum (Life Technologies, Cergy-Pontoise, France) were transfected using LipofectinÒ Reagent (Life Technol- ogies). After 3 days of culture, 500 lgÆmL )1 of neomycin (G418, Life Technologies) was added, and resistant cells, obtained after two weeks of selection, were cloned by limiting dilution. The resulting clones were tested for CCR5 cell surface expression by FACS. In order to coexpress CCR5 and CD4 on CHO cells, clones expressing CCR5 were also transfected with pcDNA3 containing the CD4 gene. This plasmid was made by removing the CD4 cDNA from the pT4b plasmid (obtained from the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH) between the EcoRI and XbaIsites, and inserting the gene between the corresponding sites into the polylinker of pcDNA3. CD4-his was made by cutting a CD4–His 6 construct (kindly donated by M. Marsh, Medical Research Council Laboratory, University College, London, UK) between two BamHI sites from pSG5 and recloning the gene into the polylinker of pcDNA3. After 2 weeks of culture in the presence of G418, CD4 positive cells were isolated using magnetic beads bearing an anti-CD4 Ig (DynabeadsÒ M-450 CD4, Dynal). After two rounds of selection, cells were cloned by limiting dilution and the clones analysed by FACS for coexpression of CCR5 and CD4. Western blot analysis and 35 S-labelled CCR5-myc-his detection CCR5-myc-his containing samples were mixed 1 : 1 with Laemmli sample buffer and loaded on SDS/PAGE gels without heating. After migration, proteins were transferred to nitrocellulose membranes, probed with the anti-myc mAb 9E10 (Sigma) and revealed with horseradish peroxi- dase-conjugated goat anti-mouse IgG and the NEN signal chemiluminescence kit (NEN Life Science Products). For 35 S-labelled CCR5-myc-his detection, SDS/PAGE gels were dried in a gel dryer, and exposed for 5–20 h on an Molecular Dynamics (Bondoufle, France) phosphor screen. The screen was scanned with a STORM Ò device anddatawereanalysedwith IQNT software (Molecular Dynamics). CCR5-myc-his labelling and purification CHO cells (grown to 90% confluence in 140 mm dishes) were washed twice with NaCl/P i and incubated with DMEM without methionine and cysteine plus 4% dialysed foetal calf serum for 6 h. A 35 S-labelled methionine/cysteine mixture (Easytag, New England Nuclear, Zaventem, Belgium) was added (250 lCi per dish). After overnight incubation at 37 °C, about 200 millions cells were removed from Petri dishes with NaCl/P i , containing 10 m M EDTA, andwashedwithNaCl/P i . After centrifugation, the pellet was suspended in lysis buffer [NaCl/P i supplemented with 1% Triton X-100, 1000 UÆmL )1 DNAse I, 1 m M MgCl 2 , and the protease inhibitors Chymostatin, Leupeptin, Antipaı ¨ n, Pepstatin (Sigma), all at 1 lgÆmL )1 ]. After 45 min at 4 °C, the preparation was centrifuged at 25 000 g for 15 min. CCR5-myc-his was purified from the supernatant by immobilized metal ion affinity chromatogra- phy (Ni-nitrilotriacetic acid, Qiagen, Courtaboeuf, France) or anti-polyhistidine Igs immobilized on agarose beads (Sigma). In both cases, N-dodecyl-b- D -maltopyranoside (DDM) was used at 0.3 m M final concentration in all the buffers needed for the various purification steps, thereby replacing the Triton X-100 of the lysis buffer in the purified 5164 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 product. For purification on the Ni-nitrilotriacetic acid column, the supernatant was diluted with an equal volume of binding buffer (Tris 20 m M pH 8, NaCl 500 m M ,imidazole 5m M , DDM 0.3 m M ), and passed twice through the column. The column was then washed with Tris 20 m M ,NaCl 500 m M ,imidazole25m M , DDM 0.3 m M ,pH8andeluted with a similar buffer containing 200 m M imidazole. For purification on anti-polyhistidine agarose, the supernatant was passed through the column which was washed with Hepes 2.5 m M pH 7.4, NaCl 145 m M ,DDM0.3m M . CCR5-myc-his was eluted with the same buffer, to which imidazole (200 m M ) was added. Liposome preparation and CCR5-myc-his reconstitution Large unilamellar liposomes (0.1 lm in diameter) were prepared from dry lipid films containing egg-PtdCho (55 mol%), egg-PtdEth (27 mol%), SM (8.2 mol%), cholesterol (8.2 mol%), N-Rh-PtdEth (0.82 mol%), N-NBD-PtdEth (0.82 mol%) as described previously [34]. For CCR5-myc-his reconstitution, these liposomes (66 l M of phospholipids) were first incubated with DDM at 0.2 m M for 30 min at 4 °C. Purified fractions of CCR5-myc-his in DDM (0.3 m M )werethenmixedwith detergent saturated liposomes at a 1 : 3 vol. ratio of protein preparation to liposomes. After a 30-min incu- bation, detergent was removed by the addition of Biobeads SM-2 (Bio-Rad). A 4-h incubation with 0.5 mg of beads was followed by two 1-h incubations with an additional 10 mg of beads with continuous agitation at 4 °C. After detergent extraction, the resulting proteoliposomes were pelleted three times at 100 000 g for 30 min. All steps were at 4 °C. MIP-1a binding experiments CHO cells (1 · 10 6 cells in 50 lL) were suspended in Hepes 50 m M pH 7.4, MgCl 2 5m M ,CaCl 2 1m M , BSA 0.5% at 25 °C for 1 h in the presence of 0.55 n M [ 125 I]MIP-1a. Non– specific interactions were measured by adding an excess of unlabeled MIP-1a (500-fold). For MIP-1a binding to reconstituted CCR5-myc-his, freshly prepared proteolipo- somes (3 nmol of phospholipids) were incubated for 45 min at 25 °C(Hepes2.5 m M , NaCl 145 m M ,BSA0.1mgÆmL )1 ) in the presence of different concentrations of [ 125 I]MIP-1a andcoldMIP-1a or RANTES (1000 and 500-fold [ 125 I]MIP-1a quantities). Unbound ligand was removed by filtration on BSA-presaturated GFB-filters (Whatman), and the filters were washed twice with Hepes 2.5 m M ,NaCl 150 m M ,BSA0.1 mgÆmL )1 . The filters were then counted in a c-counter (Packard). Anti-CCR5 binding experiments Freshly prepared proteoliposomes (8 nmol of phospho- lipids) were incubated for 45 min at 23 °C in Hepes 2.5 m M pH 7.4, NaCl 145 m M ,BSA0.1mgÆmL )1 containing 10 lgÆmL of anti-CCR5 mAb 2D7 (Pharmingen), 181 or 182 (R & D). The proteoliposomes were separated from unbound antibody by ultracentrifugation (100 000 g, 30 min), the pellet was resuspended in the same buffer, and incubated with 125 I-labelled anti-mouse Ig (3 lCiÆmL )1 )for30minat4°C. The proteoliposomes were then washed with the same buffer and pelleted by ultracentrifugation. Protein orientation Proteoliposomes containing CD4, approximately 20 nmol, wereincubatedinNaCl/P i for 20 min at 37 °C, with or without trypsin (0.5 gÆL )1 ). The proteoliposomes were washed once by ultracentrifugation to remove the protease, and the pellet was resuspended in gel loading buffer for Western blot analysis. The blot was revealed by the anti- CD4 mAb Q4120. Co-immunoprecipitation of proteoliposomes 4 · 10 6 paramagnetic beads with or without anti-CD4 Igs were incubated overnight at 4 °C with 20 nmol of CD4- CCR5 proteoliposomes, in Hepes 2.5 m M ,NaCl145m M , and BSA 1 mgÆmL )1 . The beads were then washed in the same buffer five times, and once in the same buffer without BSA. Finally the beads were resuspended in sample buffer SDS/PAGE for Western blot analysis. Cell-cell fusion experiments 8 · 10 4 cells expressing the gp120/gp41 of strain ADA were seeded per well (1.55 cm diameter) in 24-well plates. The following day 18 · 10 4 CHO cells were added and after 3–5 h at 37 °C, a fusion percentage FI was calculated as FI ¼ [1 ) (number of cells/number of nuclei)] · 100. A minimum of 500 nuclei were counted for each assay. Binding of a soluble version of the JR-FL gp120/gp41 to proteoliposomes A 35 S-labelled preparation of this protein, secreted by CHO JR-FL gp160, clone A19, cells was prepared by radiolabel- ling as described above for CCR5-myc-his. Tissue culture supernatant was passed 5–8 times through a 1-mL column of Galanthus nivalis lectin coupled to Sepharose beads equilibrated with Hepes 2.5 m M pH 7.4, NaCl 145 m M buffer, at approximately 1 mLÆmin )1 .Thecolumnwasthen washed with 40 vol. of Hepes 2.5 m M pH 7.4, NaCl 145 m M , and the protein was eluted with 10 vol. of the same buffer containing 250 m M a-methyl-mannopyrano- side. The protein was then concentrated by size filtration (Centricon-30, Qiagen). For binding experiments, the labelled protein (10 n M ) was added to proteoliposomes (100 l M phospholipid) in Hepes 2.5 m M pH 7.4, NaCl 145 m M ,BSA0.1%,CaCl 2 1 l M ,MgCl 2 1m M . Binding of proteoliposomes to gp120/gp41 expressing cells 200 · 10 3 ADA cells per well were seeded in 24-well plates. After two days of growth, the cells were washed twice with cold HBSS (Hank’s Balanced Saline Solution, Life Tech- nologies, Cergy-Pontoise, France) and kept for 30 min at 4 °C. About 15 nmol of proteoliposomes were diluted in 200 lL of HBSS, and incubated with the cells for 30 min at 4 °C. The cells were then washed with HBSS to remove unbound proteoliposomes, and incubated for 30 min at 37 °C. After this step, the cells were scraped from the wells Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5165 in NaCl/P i and the fluorescence associated with the cells was analysed by FACS. RESULTS Functional expression of a Myc-his tagged version of CCR5, and a his-tagged version of CD4 Because the post-translational modifications of CCR5, such as tyrosine sulfation appear to be important for its function [35], we produced the protein in mammalian cells. In order to facilitate the proteins’ purification, a recombinant version was made containing a C-terminal myc tag followed by a His 6 sequence, by cloning the CCR5 gene into a pcDNA3.1 myc-his vector, and transfecting CHO cells with this construct. After G418 selection, clones expressing CCR5- myc-his were obtained by limiting dilution and analysed by FACS using the antibody 2D7 (Fig. 1), which recognizes CCR5 [36]. Western blot analysis of the cloned protein from a postnuclear supernatant of lysed cells was carried out using the 9E10 mAb directed against the myc epitope (none of the commercially available anti-CCR5 mAbs were able to recognize wild type- or CCR5-myc-his on blots). Two proteins were detected by 9E10, one with a molecular mass of around 38 kDa, close to the estimated molecular mass of CCR5-myc-his, and a second of 33 kDa (Fig. 1). The latter protein was also detected in untransfected cells. Thus, the tagged version of CCR5 was present on the surface of transfected cells, and had the expected molecular mass. To test whether the presence of the myc-his tag at the C-terminus of CCR5 would interfere with the protein’s ability to function as a chemokine receptor or HIV coreceptor, we first determined the binding of MIP-1a to the CCR5-myc-his expressing cells. Cells expressing CCR5 or CCR5-myc-his were incubated with [ 125 I]MIP-1a alone or in the presence of a large excess of cold MIP-1a. Specific binding of the ligand to both cell types was observed (Fig. 2A). Some binding of radioactive MIP-1a to non- transfected CHO cells, even in the presence of excess cold MIP-1a was consistently observed, and probably reflects low affinity binding to heparan-like glycosaminoglycans [37]. As the myc-his tag of the protein was present at the C-terminus of the receptor which is located on the inside of the cell, we then determined if signal transduction by the protein was affected by this modification. One of the consequences of signalling is a down-modulation of the expression of CCR5 [38]. CCR5-myc-his or wtCCR5 expressing cells were incubated at 37 °Cfor30minwith 10 n M of MIP-1a and then the CCR5 present on the plasma membrane was quantified by FACS analysis using the 2D7 antibody (Fig. 2B). It was found that the 60–70% of the myc-his tagged protein was internalized, as much as for wild-type CCR5 in CHO cells [39]. To test the ability of CCR5-myc-his to function as a coreceptor for gp120/gp41 induced fusion, CHO clones coexpressing CCR5-myc-his or wt CCR5 were transfected with pcDNA3.1 containing a His 6 -tagged version of CD4, CD4-his, as described in Materials and methods. After cloning and selection by FACS analysis (not shown) of cells coexpressing CCR5 and CD4,sincytium formation with cells expressing gp120/gp41 from strain ADA was tested. The level of fusion obtained with cells expressing tagged or untagged proteins was comparable (Fig. 2C). Thus, these results indicated that CCR5-myc-his and CD4-his are functional HIV coreceptor and receptors, respectively. Purification of CCR5-myc-his For the purification of CCR5-myc-his we tested two different methods based on the presence of the six histidines at the C-terminus of the protein. The first involved immobilized metal ion affinity chromatography. Cells were lysed with a buffer containing Triton X-100 and spun at 25 000 g for 15 min. The supernatant was applied to a Nickel-nitrilotriacetic acid (Ni-nitrilotriacetic acid) column. The column was washed with different buffers to remove contaminant proteins and then eluted with a buffer containing 200 m M imidazole and the detergent, as des- cribed in Materials and methods. The protein was followed throughout the different purification steps by Western blot, using the anti-myc Ig, and by SDS/PAGE of 35 S-labelled protein (Fig. 3A). CCR5 was quantitatively retained on the column, and could be eluted with imidazole. The protein was unidentifiable by autoradiography in cell lysates, but appeared as the predominant band after purification, although a background of contamination by other proteins was still present. The second method involved His1 anti-His 6 Igs coupled to Sepharose beads. A column made of this material also retained CCR5 quantitatively, and elution with 200 m M imidazole resulted in a more highly purified protein than purification on Ni-nitrilo- triacetic acid columns (Fig. 3). Ten percent of the total radioactivity present in the lane containing CCR5-myc-his purified on Ni-nitrilotriacetic acid columns was associated with the protein, whereas 36% of the total radioactivity was associated with CCR5-myc-his after purification on an antihistidine column. Reconstitution of CCR5-myc-his A limited number of protocols for the membrane reconsti- tution of GPCR exist, and there does not seem to be a Fig. 1. CCR5-myc-his expression in CHO cells. CHO cells were transfected with pcDNA3 containing the ccr5-myc-his gene. Expres- sion was measured by FACS, using the 2D7 anti-CCR5 Ig [36]. Western blot analysis was carried out using an anti-myc Ig. Trans- fected cells are marked with +, nontransfected cells that were used as a negative control, with The approximate position of molecular mass markers (in kDa) is shown on the right. 5166 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 generally applicable method. Several coinsertion reconsti- tution methods, involving the preparation of a mixture of detergent, lipids and protein, followed by removal of detergent, were first tried for CCR5. CCR5-myc-his expressing cells were lysed with Triton X-100, and the protein was immobilized on Ni-nitrilotriacetic acid columns. To exchange the Triton X-100 for other detergents, the column was then washed with buffer containing another detergent, imidazole elution was performed in this detergent and the eluate applied to dry lipid films. Several detergents with a high critical micelle concentration (cmc), such as b-D-octylglucoside and 3-[(3-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate (CHAPS) were tested, and detergent removal methods tested involved rapid dilution, dialysis or gel filtration. The low cmc detergents octaethye- leneglycol-mono-N-dodecylether (C 12 E 8 ) and Triton X-100 were also tested. In these cases, detergent was removed by hydrophobic polystyrene beads (Biobeads SM2). We were unable to reconstitute the CCR5-myc-his in a lipid bilayer by any of these methods. Postinsertion reconstitution was therefore attempted next. This involves adding the detergent-solubilized protein to preformed membranes, followed by detergent removal. Although the physicochemical basis of this type of recon- stitution is not clear, saturating the membranes with detergent before protein addition was found to promote reconstitution in a number of cases [1]. One detergent, which has been used frequently for postinsertion reconstitution, is DDM. This detergent, like other alkyl-glucoside detergents, also seems to conserve the structure of solubilized CCR5 [40]. We first determined how much DDM could be added to liposomes without solubilizing them. To this end, liposomes containing the lipids egg-PtdCho (55 mol%), egg-PtdEth (27 mol%), SM (8.2 mol%), cholesterol (8.2 mol%), and the fluorescent phospholipid analogues N-NBD-PtdEth and N-Rh-PtdEth (0.82 mol% each) were produced. Resonance energy transfer (RET) between these probes depends on their membrane concentration; insertion of detergent into the membrane should result in a gradual decrease of RET [41,42], and lysis of the liposomes in abrupt abolition of RET. A decrease in RET results in an increase in NBD fluorescence [43]. Addition of DDM at concentra- tions between 0.1 and 0.4 m M to 1.5 mL of a 66-l M (lipid concentration) solution of liposomes at 4 °C led to gradual increases in NBD fluorescence, which reached stable levels after about 30 min (Fig. 4A). In contrast, addition of Triton X-100 (0.5% w/v) at the end of the experiment led to an immediate jump in fluorescence, indicating the lysis of the membranes. The cmc of DDM is around 0.25 m M .Thirty- five percent fluorescence dequenching was obtained at 0.2 m M DDM. Assuming that DDM occupies about half as much volume in the membrane as a phospholipid and that detergent is only incorporated in the outer leaflet, the Fig. 2. The tagged version of CCR5 binds MIP-1a, is down-regulated and can serve as a coreceptor for fusion. A: CHO cells expressing CCR5-myc-his, wt CCR5 or CHO cells transfected with pcDNA3 without the ccr5-myc-his gene as a negative control were incubated with 0.55 n M [ 125 I]MIP-1a or 0.55 n M [ 125 I]MIP-1a in the presence of 250 n M of cold MIP-1a in Hepes 50 m M pH 7.4, MgCl 2 5m M ,CaCl 2 1m M , BSA 0.5% at room temperature. The cells were washed twice with Hepes 10 m M pH 7.4, NaCl 0.5 M , BSA 0.5%. Specific binding was then calculated by subtracting the cell-associated counts in the presence of cold MIP-1a from those obtained with radioactive MIP-1a only. B: Downregulation of CCR5 after MIP-1a stimulation. Cells were incubated with (open bars) or without (closed bars) 10 n M MIP- 1a for 30 min at 37 °C, and then CCR5 expression on the cell surface was measured by flow cytometry using 5 lgÆmL )1 of antibody 2D7 and an FITC-labelled secondary antibody. C: Fusion between cells expressing different versions of CCR5, or nontransfected cells (CHO) with HeLa cells expressing gp120/gp41 from strain ADA. 8 · 10 5 ADA cells were plated on 35 mm Petri dishes, overlaid with 1.6 · 10 6 CHO cells the following day and incubated for 3 h at 37 °C. A fusion index was calculated as described before [55]. Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5167 detergent to phospholipid ratio in this leaflet is 1 : 2, close to the optimal conditions for reconstitution [1,44]. To reconstitute CCR5, we then incubated liposomes (66 l M ) with 0.2 m M of detergent. After reaching a stable level of fluorescence, one-quarter volume of CCR5-myc-his, purified on a Ni-nitrilotriacetic acid column in 0.3 m M of DDM, the minimum concentration required to solubilize the protein, was added (Fig. 4B). After 30 min of coincu- bation at 4 °C, detergent was removed. In postinsertion reconstitution, usually some detergent is slowly removed first, with a low amount of hydrophobic biobeads SM2, and then the rest is removed rapidly by incubation with larger amounts of beads [1]. For initial slow removal, several initial bead concentrations (from 0.25 to 1.5 mg per mL) and various incubation times (from 2.5 to 5 h) were tried. For the second step, two additions of 2.5–20 mg per mL for 45 min to 1 h were tested. The final protocol involved a 4-h incubation with 0.5 mg of beads, followed by the addition of 5 mg of beads, per mL of reconstitution solution. After 1 h, these beads were removed from the mixture, and 10 mg of fresh beads was added for another hour of incubation at 4 °C. The mixture was stirred continuously. The removal of detergent was followed by monitoring the NBD fluores- cence (Fig. 4B). The resulting proteoliposomes were pelleted by ultracentrifugation, resuspended in detergent free buffer, pelleted again, and this step was then repeated. Using this protocol, approximately 12% of the purified CCR5 (approximately 3% of the total detergent-solubilized CCR5) was found in the final pellet, whereas unincorpo- rated protein was found in the first supernatant (Fig. 5). Under these conditions, more than 65% of the total proteins incorporated in the proteoliposomes was CCR5. If deter- gent was removed from protein in the absence of liposomes by this protocol, no protein was pelleted. Thus, the proteins present in the pellet were associated with the liposomes. Moreover, as judged by the fluorescence quenching of N-NBD-PtdEth by N-Rh-PtdEth in the pelleted proteo- liposomes, the first centrifugation step also removed the remaining detergent from the proteoliposomes (Fig. 4B). Similar results were obtained with CCR5-myc-his protein Fig. 3. CCR5-myc-his purification. CCR5-myc-his was purified by immobilized metal ion affinity (A) or immunoaffinity chromatography using anti-histidine Igs (B). Results were analysed by Western blot as in Fig. 1, or by autoradiography of 35 S-labelled proteins. Fig. 4. Reconstitution of CCR5-myc-his. A: Saturation of liposomes with DDM. DDM, at final concentrations between 0.1 and 0.4 m M was added to liposomes (66 l M of lipid) containing N-NBD-PtdEth and N-Rh-PtdEth at time 0, and the fluorescence of N-NBD-PtdEth was measured and normalized to a scale where 0% representing the initial residual fluorescence of N-NBD-PtdEth before the addition of detergent, and 100% represents the fluorescence of completely dequenched N-NBD-PtdEth, obtained by addition of Triton X-100 (0.5% w/v), corrected for the quenching of NBD by Triton [43] (marked with ÔTXÕ). Note the rapid increase after addition of Triton X-100, and the lack of interaction at DDM concentrations below its cmc. B: Reconstitution. At time 0, 0.2 m M of DDM was added to liposomes as described above, then protein was added in 0.3 m M of DDM (P), and detergent was removed by addition of 0.5 mg Biobeads SM-2, followed by a second addition of 10 mg of beads. These beads were then removed from the mixture, and 10 mg of fresh beads were added (indicated by Ô0.5Õ, Ô10Õ and Ô10Õ). All incubations were at 4 °C. Pellet denotes the fluorescence of the same quantity of lipid after purification by ultracentrifugation as described in the text. 5168 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 purified on antihistidine coupled agarose beads. In conclu- sion, postinsertion reconstitution produced CCR5 contain- ing liposomes without residual detergent. Characterization of reconstituted CCR5-myc-his To characterize the membrane orientation and functionality of the reconstituted protein, we first measured the binding of antibody 2D7, 181 or 182, which recognize the extracellular loops of CCR5 [36,45], to the proteoliposomes. The proteoliposomes were incubated with the anti-CCR5 anti- body, pelleted by ultracentrifugation, resuspended, incuba- ted with a 125 I-labelled secondary antibody, and washed by ultracentrifugation. Specific binding of monoclonal anti- CCR5 to CCR5-myc-his proteoliposomes could be demon- strated (Fig. 6). These data demonstrated that at least some of the CCR5 is oriented with its N-terminus toward the outside of the liposomes and the binding of antibody 2D7, known to recognize a conformational epitope on CCR5 [36], indicated that the conformation of the receptor was maintained in the liposomal membrane. Binding of [ 125 I]MIP-1a to the proteoliposomes was then measured. Proteoliposomes were incubated with the ligand for 45 min at room temperature, and subsequently filtered on BSA-presaturated GF-B filters, which were repeatedly washed, after which the filters were counted. Specific binding of [ 125 I]MIP-1a to CCR5-myc-his liposomes was demonstrated. However, when we attempted to measure the number of binding sites per proteoliposome by adding mixtures of [ 125 I]MIP-1a and increasing concentrations of cold MIP-1a, a strange phenomenon was observed; addi- tion of cold MIP-1a strongly increased the binding of [ 125 I]MIP-1a, in fairly linear manner (Fig. 7). Although we have no explanation for this behaviour, nonclassical binding of MIP-1a has been described before [16]. We therefore attempted to use another ligand, RANTES, to compete with MIP-1a for specific binding sites on proteoliposomes; partial competition by RANTES and concentration- dependent specific radioactive MIP-1a binding to proteo- liposomes were observed (Fig. 8). However, at the highest concentration of radioactive MIP-1a attainable (4 n M ), we did not reach saturation, which could indicate the presence of a high concentration of CCR5 in the membranes. In conclusion, these experiments demonstrate that at least part of the CCR5 present in proteoliposomes is correctly oriented and still capable of recognizing its natural ligand. Co-reconstitution of CD4 and CCR5 in the same membrane To preserve potential associations between CD4-his and CCR5-myc-his, these two proteins were then copurified from the same cell line on a Ni-nitrilotriacetic acid column; as they carried the same hexahistidine tags, optimal conditions for their elution were nearly identical. We then attempted to reconstitute the two proteins together in the Fig. 7. Non-classical behaviour of MIP-1a observed with CCR5 pro- teoliposomes. Proteoliposomes (6 nmol of phospholipids) were incu- batedwith[ 125 I]MIP-1a (open circles) or a mixture of radioactive plus a 500-fold excess of cold MIP-1a (closed squares) in Hepes 2.5 m M pH 7.4, NaCl 145 m M ,BSA0.1mgÆmL )1 buffer for 45 min at room temperature, washed on GFB-Whatman filters with the same buffer and then the filters were counted. Fig. 6. Specific binding of anti-CCR5 antibody to proteoliposomes. Proteoliposomes (8 nmol of phospholipids) were incubated for 30 min with 10 lg/mL of antibody 2D7, 181 or 182, washed once with Hepes 2.5 m M pH 7.4, NaCl 145 m M ,BSA0.1 mgÆmL )1 , and then incubated with 125 I-labelled sheep anti-mouse IgG (5 lCiÆmL )1 )for30minat room temperature. The proteoliposomes were then washed twice in the same buffer and bound radioactivity was determined. The negative control consists of incubating the proteoliposomes with the labelled secondary antibody only. Fig. 5. Analysis of CCR5-myc-his reconstituted into liposomes. CCR5- myc-his proteoliposomes were washed three times by ultracentrifu- gation. The consecutive supernatants S1, S2 and S3 and the final proteoliposome pellet (PL) were analysed by Western blot and auto- radiography, and show successful reconstitution of the protein, as well as the absence of unincorporated protein in the second and third supernatant. Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5169 same liposomal membrane, simply by applying the protocol worked out for CCR5-myc-his, in spite of the biochemical differences between the two proteins. To our surprise, Western blot analysis readily demonstrated the presence of CD4-his and CCR5-myc-his in the proteoliposomal pellet, indicating that CD4-his could be reconstituted using a protocol optimized for a RCPG (Fig. 9, Panel A). However, the proteins could still be present in individual liposomes. To determine whether CD4 and CCR5 were present together in individual or separate liposomes, these were immunoprecipitated with paramagnetic beads bearing anti- CD4 Igs. The precipitate analysed by Western blot with an anti-myc Ig recognizing the myc-his tagged CCR5. As showninFig.9(PanelB),CCR5-myc-his(the38kDa band) was found to be present in these proteoliposomes. Thus, CD4 and CCR5 were present together in individual liposomal membranes. The relative stoechiometry of the two incorporated proteins was estimated by Western blot analysis. If we assume that the anti-His 6 Ig used (anti-tetra- his Ig, QIAGEN) recognizes both His 6 -tagged proteins with similar affinities, quantifying the relative intensities of each bandwouldleadtoanestimateratioof5:1forthe CD4-CCR5. In order to determine the orientation of CD4 with respect to the proteoliposomal membranes, proteoliposomes were digested with trypsin, after which the trypsin was removed, and the CD4 quantified by Western blot relative to Fig. 9. Co-reconstitution of CD4 and CCR5. A: Western blot showing the presence of CD4 and CCR5, after purification by immobilized metal ion affinity from a lysate obtained from CHO cells, coexpressing CD4-his and CCR5-myc-his and reconstitution by the protocol worked out for CCR5-myc-his. S1, S2 and S3 represent the three consecutive supernatants obtained after ultracentrifugation, and PL the final pellet (cf. Fig. 5). The Q4120 antibody (2 lgÆmL )1 )wasused for detection of CD4, and an anti-myc Ig for CCR5, as described in the legend of Fig. 1. B: 20 nmol of proteoliposomes containing CD4 and CCR5 were incubated with 4 · 10 6 paramagnetic beads with (lane A) or without (lane B) the anti-CD4 Ig. After washing the beads they were resuspended in gel electrophoresis sample buffer and analysed by Western blot with the anti-myc Ig. Besides the 38 kDa band corres- ponding to CCR5-myc-his which is present in lane a, the heavy and light chains of the anti-CD4 Ig (50 and 25 kDa, respectively, lane a) are also revealed by the anti-mouse IgG secondary antibody, while no CCR5isdetectedinlaneb. Fig. 8. MIP-1a binds specifically to CCR5 proteoliposomes. CCR5- myc-his proteoliposomes (6 nmol of phospholipids) were incubated with [ 125 I]MIP-1a in Hepes 2.5 m M pH 7.4, NaCl 145 m M ,BSA 0.1 mgÆmL )1 for 45 min at room temperature, washed on GFB- Whatman filters with the same buffer and then the filters were counted. A: Binding in the presence of radioactive MIP-1a only, or in the presence of a 500-fold excess of nonradioactive RANTES. B: Specific binding at different concentrations of MIP-1a to CCR5-myc-his liposomes. The proteoliposome-associated counts in the presence of a 500-fold excess of cold RANTES were subtracted. 5170 F. Devesa et al. (Eur. J. Biochem. 269) Ó FEBS 2002 untreated controls. After digestion, no more CD4 was detected by Western blot using the anti-CD4 mAb Q4120, suggesting that the vast majority of CD4 was oriented right- side out, as the ectodomain of the protein is recognized by the antibody. In order to test the biological activity of CD4, a 35 S-labelled soluble construct, containing the whole ectodo- main of the gp120/gp41 of strain JR-FL was produced and purified over a lectin column as described in Materials and methods. It was found that this protein bound specifically to CCR5/CD4 liposomes. As previously described for the binding of gp120/gp41 to CXCR4-paramagnetic proteo- liposomes [46], approximately 30% of binding was dis- placed by a 200-fold excess of cold gp120/gp41 (Fig. 10, Panel A). To determine whether the proteoliposomes could recognize the complete gp120/gp41 protein, they were incubated with cells expressing this glycoprotein complex and the fluorescence of cell-associated proteoliposomes was quantified by FACS analysis (Fig. 10, Panel B). The gp120/ gp41 expressing cells were found to bind proteoliposomes containing CCR5 and CD4 specifically. These data suggest that CD4 was also inserted right side out into proteolipo- somal membranes, and that CD4 reconstituted in those model membranes remains functional. DISCUSSION Postinsertion or coinsertion protocols have been used for the reconstitution of membrane proteins [1,47]. Co-insertion involves the removal of detergent from mixtures of free and micellar detergent and mixed protein/detergent and lipid/ detergent micelles. Upon removal of detergent, the protein will likely become insoluble and either self-aggregate, or associate with lipids if these also become insoluble at this point. In the latter case, a membrane containing the protein may be formed. However, protein aggregates may be formed on the one hand and lipid membranes with little incorporated protein on the other [48]. The success of this method thus probably depends on the relative affinities of interaction of the protein and lipid with the detergent. As it is not always possible to predict these parameters, a variety of detergents and detergent removal protocols have to be tested for every individual protein, and successful reconstitutions by these methods have involved a variety of lipid/detergent combinations and detergent removal protocols [3,5,6]. Postinsertion reconstitution involves mixing detergent- solubilized protein with detergent-saturated liposomes, followed by detergent removal. The physicochemical basis of postinsertion reconstitution is not clear, but whether proteins will aggregate or associate with the lipid bilayer probably also depends on the relative affinities of the protein for a membrane or itself. Most successful reconsti- tutions by this method were with transporter proteins, over- expressed in bacteria, containing 6–12 transmembrane a helices [2,9–11,49]. These proteins are robust and have large hydrophobic domains, facilitating reconstitution, and their production can easily be scaled up, making the efficiency of reconstitution less critical. No GPCR over-expressed in mammalian cells had previously been reconstituted by postinsertion protocols, although some had been successfully reconstituted by coinsertion methods [4,15,17,50]. CCR5 and the related CXCR4, isolated from mammalian cells in which they were expressed at relatively high levels, were recently reconstitu- ted by an innovative method involving paramagnetic beads [16,46]. After extraction with a detergent, the proteins were immunoprecipitated with antibodies coupled to the beads and then lipid membranes were formed around these. An advantage of this method is that the antibodies can orient the GPCR. However, the immobilization on beads may affect the protein’s behaviour. In order to develop a postinsertion reconstitution proto- col, the choice of the detergent is crucial. DDM, b-D-octylglucoside or Triton X-100 are among the deter- gents which seems most suited for this type of reconstitution [11,49]. Interestingly, both CD4 and CCR5 are associated with membrane raft microdomains [51,52], known to be solubilized preferentially by alkyl-glucoside type detergents [53]. As the native conformation of CCR5 was well preserved in DDM [40], probably because of its structural Fig. 10. Binding of soluble gp 120 or cells expressing gp120/gp41 to proteoliposomes containing CD4 and CCR5. A: Binding of the 35 S-labelled soluble version of gp120/gp41, strain JR-FL to CD4- CCR5 or only CCR5 containing proteoliposomes containing CD4 and CCR5, in the absence or presence of a 200-fold excess (Ô200xÕ)ofcold protein, as described in Materials and methods. Error bars are one standard deviation. B: FACS analysis of cells expressing gp120/gp41 (ADA strain), incubated with about 15 nmol of proteoliposomes (PL, thick line) or liposomes (L, thick line). The thin line corresponds to the auto-fluorescence of the cells. Ó FEBS 2002 CCR5 and CD4 reconstitution (Eur. J. Biochem. 269) 5171 resemblance to raft glycolipids, this detergent was chosen. Efficient reconstitution is strongly dependent on detergent concentration for a given lipid/protein ratio [44]. The optimal detergent concentration was frequently found to be close to that at the onset of liposome solubilization [2], and the most critical steps in postinsertion usually are the (stepwise) addition of the detergent to the membranes without causing their solubilization, followed by detergent removal [1]. Saturation of liposomes with detergent is mostly measured by techniques [1,54] that require high lipid concentrations and do not give quantitative information on the concentration of detergent present in the membrane. As expression of proteins in mammalian cells leads to the isolation of lg rather than mg of receptors, small quantities of liposomes can be produced only. In this paper, detergent insertion was therefore followed by resonance energy transfer measurements using fluorescent phospholipid ana- logues present in the liposomal membrane [41,42]. Satura- tion with 0.2 m M of DDM was found to suffice to destabilize membranes for reconstitution, leading to a bulk solution lipid/detergent molecular ratio of about 3 : 1 in solution. Usually, at higher lipid concentrations, a 1 : 1 ratio is attained [10]. Besides differences in the nature of the detergent, this could be a consequence of the lipid concen- tration, because the detergent partitions between the solution, protein and membrane, but only the concentration in the membrane determines the outcome of reconstitution. Therefore, an advantage of the resonance energy transfer method is that the membrane concentration of the detergent can be estimated. At less than 0.2 m M DDM, poor reconstitution of proteins was observed, and at higher concentrations liposomes were solubilized. In the latter case, if detergent was removed with beads, no reconstitution of protein was observed at all. The resonance energy transfer assay also allowed direct observation of the removal of detergent from the membrane in real time, and therefore the efficient comparison of a number of protocols. Initial slow detergent removal and biobeads quantity were found to be crucial for the recovery of high concentrations of functional proteins in proteoliposomes. An initial four-hour incuba- tion time with 0.25 mg of biobeads per ml led to optimal protein incorporation. Studies of molecular interactions between CD4 and CCR5 reconstituted in proteoliposomes could lead to an understanding of the constitutive associ- ation of CCR5 and CD4 in vivo, recently suggested to be important for HIV entry [33]. 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XbaIsites, and inserting the gene between the corresponding sites into the polylinker of pcDNA3. CD4- his was made by cutting a CD4 His 6 construct (kindly donated by