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Structural basis of coreceptor recognition by HIV-1 envelope spike


HIV-1 envelope glycoprotein (Env), which consists of trimeric (gp160)3 cleaved to (gp120 and gp41)3, interacts with the primary receptor CD4 and a coreceptor (such as chemokine receptor CCR5) to fuse viral and target-cell membranes. The gp120–coreceptor interaction has previously been proposed as the most crucial trigger for unleashing the fusogenic potential of gp41. Here we report a cryo-electron microscopy structure of a full-length gp120 in complex with soluble CD4 and unmodified human CCR5, at 3.9 Å resolution. The V3 loop of gp120 inserts into the chemokine-binding pocket formed by seven transmembrane helices of CCR5, and the N terminus of CCR5 contacts the CD4-induced bridging sheet of gp120. CCR5 induces no obvious allosteric changes in gp120 that can propagate to gp41; it does bring the Env trimer close to the target membrane. The N terminus of gp120, which is gripped by gp41 in the pre-fusion or CD4-bound Env, flips back in the CCR5-bound conformation and may irreversibly destabilize gp41 to initiate fusion. The coreceptor probably functions by stabilizing and anchoring the CD4-induced conformation of Env near the cell membrane. These results advance our understanding of HIV-1 entry into host cells and may guide the development of vaccines and therapeutic agents.

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Data availability

The atomic structure coordinates are deposited in the RCSB Protein Data Bank (PDB) under the accession numbers 6MEO and 6MET; and the electron microscopy maps have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession numbers EMD-9108 and EMD-9109. All other related data generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


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We thank S. Harrison and A. Kruse for advice, K. Song, J. Chen, R. Martin and W. Chang for technical assistance, N. Grigorieff and T. Grant for discussion at the early stage of the project, and S. Harrison and A. Kruse for critical reading of the manuscript. This work was supported by NIH grants AI141002 (to B.C.), AI106488 (to B.C.), AI129721 (to B.C.), AI127193 (to B.C. and J.J.C.), the Center for HIV/AIDS Vaccine Immunology - Immunogen Design AI-100645 (to B. F. Haynes), and Collaboration for AIDS Vaccine Discovery (CAVD) grant OPP1169339 (to D. H. Barouch from the Bill and Melinda Gates Foundation).

Reviewer information

Nature thanks G. Melikyan, S. Subramaniam and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Author information

B.C. and M.M.S. designed the experiments. H.P. and M.M.S. purified the CD4–gp120–CCR5 complex. J.L. performed CCR5 chemokine receptor assays. M.M.S. and S.R.-V. carried out CCR5 coreceptor functional assays. M.M.S. performed electron microscopy data collection with contributions from C.X. M.L. processed the initial negative-stain data. M.M.S. processed the cryo-EM data with contributions from M.L., and built the atomic model with help from B.C. All authors analysed the data. B.C. and M.M.S. wrote the manuscript with input from M.L. and J.L.

Correspondence to Bing Chen.

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The authors declare no competing interests.

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Extended data figures and tables

Extended Data Fig. 1 Previously known structures of CCR5 and CXCR4.

CCR5 and CXCR4 were identified as the coreceptors for HIV-1 entry in 199671,72,73,74,75,76,77. a, b, Crystal structures of a modified CCR5 (C224–N226 deleted and replaced with rubredoxin; ΔF320–L352; and the point mutations C58Y, G163N, A233D and K303E) in complex with the HIV entry-inhibitor maraviroc (PDB ID: 4MBS7) (a) and a modified chemokine [5P7]CCL5 (an antagonist; PDB ID: 5UIW10) (b). CCR5 is shown in ribbon diagram in blue, with the internally fused rubredoxin in magenta and the ligands in yellow. ce, Crystal structures of an engineered CXCR4 in complex with a viral chemokine antagonist vMIP-II (PDB ID: 4RWS15) (c), a small molecule antagonist IT1t (PDB ID: 3ODU14) (d) and a cyclic peptide antagonist CVX15 (PDB ID: 3OE014) (e). CXCR4 is shown in green, the fused T4 lysozyme in magenta and the ligands in yellow.

Extended Data Fig. 2 Characterization of stable cell lines (HEK293T and Expi293F) expressing wild-type human CCR5.

a, Chemokine receptor assay. HEK293T and HEK293T-CCR5 (stable) cells were treated with different concentrations of CCL5. Ft/F0 is a fluorescence-signal ratio proportional to that of intracellular cAMP concentration at 40 min after CCL5 activation and at time 0. The dose–response curves were plotted for both HEK293T (black) and HEK293T-CCR5 (red) cells. The experiment was carried out in quadruplicate, and repeated at least three times with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel. b, Flow cytometry histograms of HIV-1 gp120 binding to CCR5 expressed on the cell surfaces in the absence (orange) or presence (red) of soluble CD4. HEK293T cells (black), CCR5-expressing cells only (grey) and CCR5-expressing cells with soluble CD4 only (blue) were negative controls. The experiment was repeated independently at least twice with similar results. c, HIV-1 Env-mediated cell–cell fusion. HEK293T cells stably transfected with CCR5 were mixed with HIV-1 Env (gp160)-expressing cells in the absence or presence of soluble CD4. The CCR5 cells fuse with CD4-triggered Env cells very efficiently, and form large syncytia that cover almost the entire well. The experiment was repeated independently twice with similar results. d, Chemokine receptor assay by various ligands. As in a, Expi293F and Expi293F-CCR5 (stable) cells were treated with CCL5, gp120, CD4 or the complex of gp120 and CD4. The dose–response curves were plotted for both Expi293F as a control (left) and Expi293F-CCR5 (right) cells, with different ligands as indicated. The experiment was carried out in quadruplicate and repeated at least three times with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel. e, Left, kinetic curves of 5 representative wells of HEK293T-CCR5 cells treated with 5 different ligands as indicated. ATP activates the endogenous Gq-coupled G-protein-coupled receptor (P2Y receptor), as a positive control. The ratio represents fluorescence intensity divided by baseline intensity. Right, dose–response curve of each ligand. The y axis is a background-subtracted ratio (peak fluorescent intensity ratio − 1). We conclude that our gp120 and gp120–CD4 do not activate G-protein-mediated calcium flux at the concentrations tested here. The experiment was carried out in quadruplicate and repeated twice with similar results. Error bars indicate the standard deviation calculated by the STDEV function in Excel.

Extended Data Fig. 3 Purification of the CD4-gp120-CCR5 complex.

a, Schematic of expression constructs for HIV-1 gp120, human CCR5 and CD4. Segments of gp120 are designated as follows: C1–C5, conserved regions 1–5; V1–V5, variable regions 1–5; and His-tag, a six-histidine tag. Tree-like symbols represent glycans. Abbreviations used for segments of CCR5 are: N, N terminus; TM1–TM7, transmembrane helices 1–7; ECL1–ECL3, extracellular loops 1–3; ICL1–ICL3, intracellular loops 1–3; and CT, cytoplasmic tail. For CD4, the following abbreviations are used: D1–D4, immunoglobulin (Ig) domains 1–4; and strep tag, a purification tag. The transmembrane segment (TM) and cytoplasmic tail (CT) in grey are truncated in the expression construct. b, Unmodified human CCR5 in complex with HIV-1 gp120 and four-domain CD4 was purified by the following steps. (1) Complex formation: HIV-1 gp120 (light blue) and strep-tagged, four-domain CD4 (green) were incubated with CCR5 (magenta)-expressing cells to allow formation of the CD4–gp120–CCR5 complex on cell surfaces. (2) Strep-tag purification: the CCR5 complex and some of the CD4–gp120 complex were captured to strep-tactin resin via the strep-tagged CD4 (strep tag in purple). They were eluted by d-desthiobiotin under mild conditions. (3) Negative selection by an anti-V3 antibody to remove the CD4–gp120 complex. The CCR5 complex was further purified by size-exclusion chromatography. c, The purified CD4–gp120–CCR5 complex was resolved by gel-filtration chromatography on a Superose 6 column in the presence of the detergent LMNG. The molecular-mass standards include thyoglobulin (670 kDa), ferritin (440 kDa), γ-globulin (158 kDa) and ovalbumin (44 kDa). The expected size of the CCR5 complex is ~310 kDa (120 kDa for gp120, 50 kDa for four-domain CD4, 40 kDa for CCR5 and ~100 kDa for LMNG micelle). Peak fractions were analysed by Coomassie-stained SDS–PAGE (lanes 1–3). Labelled bands were confirmed by western blot and protein sequencing. The experiment was repeated independently at least 15 times with similar results.

Extended Data Fig. 4 Characterization of the CD4–gp120–CCR5 complex by electron microscopy.

a, Representative image of the CD4–gp120–CCR5 complex in negative stain. The experiment was repeated independently at least 4 times with similar results. b, 2D averages of the negatively stained CD4–gp120–CCR5 complex. The box size of 2D averages is ~330 Å. c, 3D reconstruction of the negatively stained CD4–gp120–CCR5 complex, fitted with a gp120 structure containing an extended V3 loop (PDB ID: 2QAD20), four-domain CD4 (PDB ID: 1WIO) and CCR5 (PDB ID: 4MBS). d, A representative cryo-EM image of the four-domain-CD4–gp120–CCR5 complex. Scale bar, 25 nm. Five independent large datasets were collected with similar results. e, 2D averages of the cryo-EM particle images show secondary structural features for both gp120 and CCR5.

Extended Data Fig. 5 Single-particle cryo-EM analysis of the CD4–gp120–CCR5 complex.

a, Data-processing workflow for the CD4–gp120–CCR5 complex. b, 3D reconstructions of the CD4–gp120–CCR5 complex refined with no mask at an overall resolution of 4.5 Å (left), and with a mask to exclude the last two domains of CD4 at a resolution of 3.9 Å (right), are coloured according to local resolution estimated by RELION. c, The angular distribution of the cryo-EM particles used in the reconstruction is also shown in respect to both the side and top views of the electron microscopy map. d, Gold standard Fourier shell correlation curves of the unmasked and masked electron microscopy reconstructions shown in b.

Extended Data Fig. 6 Gallery of representative density for the CD4–gp120–CCR5 complex.

Representative density in grey mesh from the 3.9 Å resolution electron microscopy map is shown for transmembrane helices TM1–TM7, the N terminus of CCR5, extracellular loop 3 (ELC3) near TM6, Tys10, Tys14 and Tyr15 (red model); two V3 regions; and for helix α1, N terminus, V3 loop, the bridging sheet and N-linked glycan at N262 of gp120 (cyan model).

Extended Data Fig. 7 Comparison of the conformations of the V3 loop and [5P7]CCL5 in complex with CCR5, as well as of gp120-bound CCR5 and G-protein-bound β2 adrenergic receptor.

a, The structures of the CD4–gp120–CCR5 and [5P7]CCL5–CCR5 complexes are superposed on CCR5 (red). The V3 loop of gp120 with its Pro311 in stick model is in cyan and [5P7]CCL5 with its Pro3 in stick model in yellow. Residues 309–316 of the V3 loop and residues 1–8 of [5P7]CCL5 adopt a very similar structure, and are highlighted in a rectangular box. b, Superposition of the structures of the N terminus of the gp120-bound CCR5 (red) and the complementarity-determining region H3 loop of antibody 412d in complex with gp120 core (green). The electron microscopy density of the CD4–gp120–CCR5 complex is shown in grey. The positions of the sulfated tyrosine (‘Tys’) residues, including Tys10 and Tys14 (from CCR5) and Tys100 and Tys100c (from 412d), are indicated. c, A model for interactions of three CD4 receptors and three CCR5 coreceptors with the SOSIP Env trimer. The side and bottom views of a composite structure of the CD4–CCR5–SOSIP Env trimer complex are shown. The model was generated using the CD4-bound SOSIP trimer (PDB ID: 5VN3) and the structure of the CD4–gp120–CCR5 complex from this study. All the structures were aligned on the basis of the core region of gp120. CCR5 is shown in red, CD4 in green, gp120 in blue, the gp120 of SOSIP in dark blue and the gp41 of SOSIP in grey. The crystallographic dimer of CCR5 (PDB ID: 4MBS) is also shown, on the left only, in a rectangular box. The observed crystallographic dimer of CCR5 or the transmembrane helix 5-mediated dimer by modelling does not seem to be relevant to binding to either monomeric or trimeric gp1207,78. d, Superposition of the structures of the gp120-bound CCR5 (red) and the Gs-protein-bound β2 adrenergic receptor (blue). The position of TM6, which is critical for the activation of G-protein-coupled receptors, is indicated.

Extended Data Fig. 8 Comparison of conformations of different structures of monomeric gp120 and various V3 loops.

a, Comparison of structures of an unliganded gp120 core (PDB ID:4OLV; purple), a CD4-bound monomeric gp120 core with the V3 loop (PDB ID: 2QAD; blue) and gp120 in complex with CD4 and CCR5 from this study (cyan). The gp120 core region is marked by a circle with a diameter of 50 Å. The N and C termini, V1V2 stem, V3 stem or loop and bridging sheet are indicated. b, Representative conformations that an HIV-1 V3 loop can adopt. From left to right, V3 loop in the unliganded SOSIP BG505 Env trimer (PDB ID: 4ZMJ); the first-V3-containing gp120 core in complex with CD4 and antibody X5 (PDB ID: 2B4C29); CD4- and 412d-bound monomeric gp120 core with V3 (PDB ID: 2QAD); CCR5-bound intact gp120 (this study); and V3 peptide in complex with antibody 447-52D (PDB ID: 3GHB36); antibody 268-D (PDB ID: 3GO137); antibody 2557 (PDB ID: 3MLV37); and antibody 10A37 (PDB ID: 5V6L38). The root-mean-square deviation of each structure (except for 5V6L), relative to the CCR5-bound gp120 monomer, is shown at the bottom in parentheses.

Extended Data Fig. 9 Model of HIV-1 Env activation to induce membrane fusion.

A hypothesis of how the cellular receptors CD4 and CCR5 trigger the HIV-1 Env trimer to induce membrane fusion and viral entry. Left, virus attaches to the target cell by gp120 (cyan) binding to CD4 (green). Helix collar (gp41), the four-helix collar gripping the N- and C termini of gp120. Right, immediate binding by CCR5 (red) prevents rapid dissociation between gp120 and CD4, stabilizes the CD4-induced conformational changes within the Env trimer and brings the trimer close to the cell membrane. Simultaneous binding of gp120 to both CD4 and CCR5 may require bending in the cell membrane. The fusion peptide (magenta) of gp41 (grey) flips out owing to intrinsic conformational dynamics, which enables the bending back of the N and C termini of gp120. This bending blocks the fusion peptide from resuming its original position in the trimer. The movements of the fusion peptide and gp120 termini effectively weaken the non-covalent association between the two subunits and may lead to partial or complete dissociation of gp120, as well as a series of refolding events in gp41 to adopt the pre-hairpin intermediate conformation (with the fusion peptides inserting into the target-cell membrane). Extended helix (gp41), three helices in the fusion-intermediate conformation of gp41.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics

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Fig. 1: Cryo-EM structure of the CD4–gp120–CCR5 complex.
Fig. 2: Interfaces between gp120 and CCR5.
Fig. 3: Conformational differences between gp120-bound CCR5 and CCR5 bound to other ligands.
Fig. 4: Conformational differences between CD4-bound and CCR5-bound gp120.
Extended Data Fig. 1: Previously known structures of CCR5 and CXCR4.
Extended Data Fig. 2: Characterization of stable cell lines (HEK293T and Expi293F) expressing wild-type human CCR5.
Extended Data Fig. 3: Purification of the CD4-gp120-CCR5 complex.
Extended Data Fig. 4: Characterization of the CD4–gp120–CCR5 complex by electron microscopy.
Extended Data Fig. 5: Single-particle cryo-EM analysis of the CD4–gp120–CCR5 complex.
Extended Data Fig. 6: Gallery of representative density for the CD4–gp120–CCR5 complex.
Extended Data Fig. 7: Comparison of the conformations of the V3 loop and [5P7]CCL5 in complex with CCR5, as well as of gp120-bound CCR5 and G-protein-bound β2 adrenergic receptor.
Extended Data Fig. 8: Comparison of conformations of different structures of monomeric gp120 and various V3 loops.
Extended Data Fig. 9: Model of HIV-1 Env activation to induce membrane fusion.


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