Research ArticleGPCR SIGNALING

CCR5 adopts three homodimeric conformations that control cell surface delivery

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Science Signaling  08 May 2018:
Vol. 11, Issue 529, eaal2869
DOI: 10.1126/scisignal.aal2869
  • Fig. 1 Cysteine cross-linking identifies TM5 and TM6 in dimer interfaces.

    (A) Schematic representation of human CCR5 (adapted from http://gpcrdb.org/protein/ccr5_human). Amino acid residues from the TM domains and helix 8 are represented. Amino acid residues substituted with a cysteine are shaded. (B) Relative cell surface expression of CCR5 cysteine mutants. HEK 293 cells transfected with plasmids encoding FLAG-WT-CCR5 or the indicated mutant receptors were stained with an antibody against the FLAG epitope and analyzed by flow cytometry. Bars represent staining efficiency for cells expressing FLAG-CCR5 mutants relative to cells expressing FLAG-WT-CCR5. Data are means ± SD of three experiments. (C to F) Disulfide cross-linking. (C and E) HEK 293 cells were transfected with plasmids encoding the indicated FLAG-CCR5 proteins. Cells were treated with (+) or without (−) CuP, lysed, and analyzed by Western blotting with an antibody against the FLAG epitope. Data are representative of three experiments. Bands that migrated at 36 kDa represent monomers, whereas those at 72 kDa represent dimers. (D and F) The percentage of dimers relative to the (monomers + dimers) amount of CCR5 was quantified by scanned densitometry analysis of the same film. Data are means ± SD of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to WT CCR5 (without or with CuP, respectively) in an unpaired, two-tailed Student’s t test.

  • Fig. 2 Lysine mutagenesis destabilizes CCR5 dimer formation.

    (A) Relative cell surface (nonpermeabilized) and total (permeabilized) expression of WT CCR5 and the indicated lysine mutants. HEK 293 cells transfected with plasmids encoding FLAG-ST-WT-CCR5 or the indicated FLAG-ST-CCR5 lysine mutants (L196K, I200K, L205K, or L208K) were permeabilized or not, stained with an antibody against FLAG, and analyzed by flow cytometry. Bars represent staining efficiency for cells expressing FLAG-ST-CCR5 mutants relative to cells expressing FLAG-ST-WT-CCR5. Data are means ± SD of three experiments. (B) Example of HTRF saturation experiments in cells coexpressing FLAG-ST-CCR5 as the donor (WT, 20 ng; L196K, 30 ng) and various amounts of FLAG-CT-CCR5 as the acceptor (WT, 0 to 200 ng; L196K, 0 to 300 ng) fitted according to a one-site binding model from one representative experiment. Data are means ± SD of four experiments. Results are expressed in milli-HTRF ratio units (mHTRF) plotted against the ratio of the amounts of cell surface donor and acceptor (ClipTb/Snap Tb, F620 nm). Dose-response curves (WT versus L196K) were compared by fitting nonlinear regression curves, and fits were compared by extra sum-of-squares F test. L196K curves were statistically significantly different compared to WT CCR5 curves. Data are means ± SD of four experiments (P < 0.001) (F2.32 = 95.21). (C) HTRF50 values (ClipTb/SnapTb values for half-maximal mHTRF) plotted as a function of the fluorescence intensity of the donor at 620 nm (F620), which represents the amount of FLAG-ST-CCR5 at the cell surface. (D) Plotted HTRF50 values for a range of F620 values from 900 to 1900 [arbitrary units (a.u.)]. **P < 0.01 and ***P < 0.001 comparing mutants to WT CCR5 in a Poisson regression test.

  • Fig. 3 Lysine mutagenesis inhibits CCR5 export from the ER.

    (A) Schematic representation of the principle of the RUSH assay. SBP-EGFP-CCR5 is retained in the ER through its interaction with streptavidin-KDEL. This interaction is mediated by the core streptavidin and the SBP (fused to CCR5). Release is induced by addition of biotin, which prevents the SBP-streptavidin interaction and enables trafficking of SBP-EGFP-CCR5 (SBP-CCR5) to the plasma membrane. (B) RUSH assay in HEK 293 cells transiently expressing the RUSH system (construct streptavidin-KDEL_EGFP-CCR5) for WT CCR5 or the indicated CCR5 lysine mutants (L196K, I200K, L205K, or L208K). The cell surface expression of CCR5 was monitored by flow cytometry after staining with an antibody against GFP at the indicated times after the addition of biotin (at time 0). Data are means ± SD of three experiments. (C) Micrographs of HEK 293 cells stably expressing FLAG-ST-WT-CCR5 or FLAG-ST-L196K transfected with plasmids expressing the ER marker DsRed-KDEL. FLAG-ST-CCR5 (WT or L196K) is shown in yellow. DsRed-KDEL is shown in blue. Scale bars, 5 μm. (D) Schematic representation of the principle of the dual RUSH assay. HEK 293 cells were cotransfected with plasmids expressing both the RUSH system for WT CCR5 (construct streptavidin-KDEL_EGFP-CCR5) and FLAG-CCR5-KKLV, which reside in the ER, at a ratio of 1:4. After the addition of biotin, the release of SBP-EGFP-CCR5 was detected as described in (A) in EGFP-positive cells. (E) Dual RUSH assay in HEK 293 cells coexpressing the RUSH system for WT CCR5 and pcDNA3 (ctl), FLAG-WT-CCR5-KKLV (WT-KKLV), FLAG-AT2R-KKLV (AT2AR-KKLV), or FLAG-CCR5–lysine mutants–KKLV (I42K-KKLV, L196K-KKLV, I200K-KKLV, L205K-KKLV, or L208K-KKLV). The cell surface expression of SBP-WT-CCR5 was monitored by flow cytometry at the indicated times after the addition of biotin (at time 0). Data are means ± SD of three experiments. **P < 0.01 and ***P < 0.001 compared to that of WT-CCR5-KKLV in an unpaired, two-tailed Student’s t test.

  • Fig. 4 MVC favors CCR5 dimerization by changing the orientation of TM5.

    (A) RUSH assay in HEK 293 cells expressing the RUSH system for L205K that were left untreated or were treated overnight with a saturating concentration (>100 nM) of MVC, 5P12, or AMD3100. The cell surface expression of SBP-L205K was monitored as described in Fig. 3B. Data are means ± SD of three experiments. (B) HTRF50 values obtained from HTRF saturation experiments performed on HEK 293 cells coexpressing FLAG-ST-WT-CCR5 and FLAG-CT-WT-CCR5 or FLAG-ST-L205K and FLAG-CT-L205K that were left untreated or were treated with 1 μM MVC overnight. Data are plotted as a function of the fluorescence intensity of the donor at 620 nm (F620). (C) Dual RUSH assay in HEK 293 cells coexpressing the RUSH system for SBP-CCR5-E283Q together with pcDNA3 (ctl), FLAG-AT2R-KKLV (ATR), FLAG-WT-CCR5-KKLV (WT), FLAG-L196K-KKLV (196), FLAG-I200K-KKLV (200), FLAG-L205K-KKLV (205), or FLAG-L208K-KKLV (208) and that were left untreated or were treated with >100 nM MVC overnight. Data are means ± SD of three experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to that of untreated conditions in an unpaired, two-tailed Student’s t test. (D) CuP or DSP cross-linking. HEK 293 cells were transfected with plasmids expressing FLAG-WT-CCR5. The cells were incubated overnight with (+) or without (−) 1 μM MVC, treated in the presence or absence of CuP or DSP, lysed, and analyzed by Western blotting with an antibody against the FLAG epitope. Data are representative of three experiments. (E) CuP-induced cross-linking of the indicated FLAG-CCR5 constructs (WT, S38C, I42C, F311C, N192C, or L205C) after the cells were left untreated or were treated with 1 μM MVC overnight. FLAG-CCR5 was detected as described in (D). Data are representative of two experiments. (F) DSP-induced cross-linking of the indicated FLAG-CCR5 constructs (WT, K59A, K138A, K171A, K197A, K219A, K228A, K229A, or K303A) after treatment with 1 μM MVC overnight. CCR5 was detected as described in (D). Data are representative of three experiments.

  • Fig. 5 3D models of unliganded CCR5 dimers.

    (A) Top: Model I5, based on the crystallographic structure of the CXCR4 homodimer. Bottom: Model I5/6, based on the crystallographic structure of the MOR homodimer. The protein backbone is represented by cylinders colored by chain (light and dark gray). Lines indicate interprotomer contacts detected during MD simulation (H-bonds, ionic bonds, aromatic interactions, and contacts between hydrophobic groups). Line thickness is proportional to the frequency of occurrence. (B) MD simulations of CCR5 dimers. Top: Model I5. Bottom: Model I5/6.Time series of rmsd values for all protein atoms using the input coordinates as reference. Data represent three independent simulations. (C) Dimer model residues predicted in the I5 (top) and I5/6 (bottom) interfaces. Side view of the initial model before simulation and not minimized. The protein backbone is represented as gray ribbons; the residues of the interface are represented with Corey, Pauling, Koltun (CPK)–colored sticks with carbon atoms in gray. Asterisks indicate residues that are positive in the next panel. (D) Boxplot of interprotomer distances during MD simulations for the I5 (top) and I5/6 models (bottom). The bottom and top of each box indicate the first and third quartiles of the distribution, respectively; the ends of the bars represent the minimum and maximum of all of the distances. A distance of 4 Å is typical for two cysteine residues in a disulfide bond (red dotted line). Blue coloring indicates direct hydrophobic contacts between a residue in the first protomer with the same residue in the second protomer.

  • Fig. 6 Effect of MVC on CCR5 structure models and dynamics.

    (A) MD simulations of the CCR5 monomer when free or in complex with MVC. Simulated systems at the end of the equilibration stage. CCR5 dimer (ribbon representation) is embedded into a lipid bilayer (line representation) surrounded by water molecules (line) and the counter ions K+ and Cl (small spheres). MVC is represented with balls (magenta). CCR5 is shown free (top) and in a complex with MVC (bottom). (B) Molecular view of residues in TM5/TM6 (top) and TM4/TM5 (bottom) (gray sticks in the free receptor and pink sticks in the complex with MVC). Dotted black lines indicate intramolecular H-bonds. TM backbones are represented by gray (free CCR5) or red (MVC-bound CCR5) ribbons. (C) Frequency of occurrence of H-bonds throughout the simulation of the free (gray) or MVC-bound (red) receptor. Top: H-bond network between the extracellular parts of TM5 and TM6. Bottom: H-bond network between the extracellular parts of TM4 and TM5. In free CCR5, the K197 amine group (NZ) was engaged in two H-bonds: one with the oxygen atom of the side chain of Q194 (OE) and one with the backbone carbonyl (O) of A159 in TM4. H-bond abbreviations, main chain: donor (N), acceptor (O); side chain: donor Q(NE), K(NZ), T(OG), N(ND); acceptor N(OD), T(OG), E(OE), Q(OE). (D) 3D model of the CCR5 dimeric state bound to MVC (IMVC), as represented in Fig. 5A. (E) Boxplot of interprotomer distances during MD simulations as described for Fig. 5D. (F) BMOE cross-linking of the indicated FLAG-tagged CCR5 constructs (WT, F193C, L196C) after overnight treatment (or not) with 1 μM MVC. FLAG-CCR5 was detected as described in Fig. 1. Data are representative of three experiments.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/529/eaal2869/DC1

    Fig. S1. Cysteine cross-linking identifies TM5 in dimer interfaces.

    Fig. S2. Lysine mutagenesis destabilizes CCR5 dimer formation without altering receptor folding.

    Fig. S3. Expression of FLAG-CCR5-KKLV.

    Fig. S4. MVC favors the transport of CCR5 to the cell surface.

    Fig. S5. MVC causes a rearrangement of CCR5 TM5.

    Fig. S6. Residues involved in interprotomer interactions.

    Fig. S7. MD simulations of the CCR5 monomer when free or in complex with MVC.

    Fig. S8. Influence of MVC on CCR5 dynamics.

    Fig. S9. Representative model of the IMVC dimer based on rigid protein-protein docking.

    Fig. S10. 3D models of the CCR5 covalent dimers obtained by cross-linking with either DSP or BMOE.

    Supplementary PDB files

  • Supplementary Materials for:

    CCR5 adopts three homodimeric conformations that control cell surface delivery

    Jun Jin, Fanny Momboisse, Gaelle Boncompain, Florian Koensgen, Zhicheng Zhou, Nelia Cordeiro, Fernando Arenzana-Seisdedos, Franck Perez, Bernard Lagane, Esther Kellenberger, Anne Brelot*

    *Corresponding author. Email: anne.brelot{at}pasteur.fr

    This PDF file includes:

    • Fig. S1. Cysteine cross-linking identifies TM5 in dimer interfaces.
    • Fig. S2. Lysine mutagenesis destabilizes CCR5 dimer formation without altering receptor folding.
    • Fig. S3. Expression of FLAG-CCR5-KKLV.
    • Fig. S4. MVC favors the transport of CCR5 to the cell surface.
    • Fig. S5. MVC causes a rearrangement of CCR5 TM5.
    • Fig. S6. Residues involved in interprotomer interactions.
    • Fig. S7. MD simulations of the CCR5 monomer when free or in complex with MVC.
    • Fig. S8. Influence of MVC on CCR5 dynamics.
    • Fig. S9. Representative model of the IMVC dimer based on rigid protein-protein docking.
    • Fig. S10. 3D models of the CCR5 covalent dimers obtained by cross-linking with either DSP or BMOE.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:


    © 2018 American Association for the Advancement of Science

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