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Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env

Nature Structural & Molecular Biology volume 22, pages 522–531 (2015)Cite this article

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Abstract

As the sole viral antigen on the HIV-1–virion surface, trimeric Env is a focus of vaccine efforts. Here we present the structure of the ligand-free HIV-1–Env trimer, fix its conformation and determine its receptor interactions. Epitope analyses revealed trimeric ligand-free Env to be structurally compatible with broadly neutralizing antibodies but not poorly neutralizing ones. We coupled these compatibility considerations with binding antigenicity to engineer conformationally fixed Envs, including a 201C 433C (DS) variant specifically recognized by broadly neutralizing antibodies. DS-Env retained nanomolar affinity for the CD4 receptor, with which it formed an asymmetric intermediate: a closed trimer bound by a single CD4 without the typical antigenic hallmarks of CD4 induction. Antigenicity-guided structural design can thus be used both to delineate mechanism and to fix conformation, with DS-Env trimers in virus-like-particle and soluble formats providing a new generation of vaccine antigens.

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Figure 1: Crystal structure of ligand-free HIV-1–Env trimer and conformational changes related to individual subunit structures and virus entry.
Figure 2: Ligand-free HIV-1–Env trimer is structurally compatible with epitopes of broadly neutralizing, but not ineffective, antibodies.
Figure 3: Structural compatibility–guided negative selection and an appropriate target antigenicity.
Figure 4: CD4-induced changes in antigenicity and conformational fixation of ligand-free HIV-1 Env.
Figure 5: Atomic-level models and physical stability of ligand-free 201C 433C mutant (DS-SOSIP).
Figure 6: DS-SOSIP binds a single CD4 without the typical antigenic hallmarks of CD4 triggering.
Figure 7: An asymmetric intermediate in the HIV-1–entry pathway.
Figure 8: A new generation of conformationally fixed HIV-1–Env trimeric immunogens.

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Acknowledgements

We thank Y. Dai (The Scripps Research Institute) for ERV MuLV Gag plasmid, M. Murphy for SPR discussions, B. Whalen (Altravax) for the Rev plasmid, members of the Structural Biology Section and Structural Bioinformatics Core, Vaccine Research Center for discussions and comments on the manuscript and the Weill Cornell Medical College, the Academic Medical Center of the University of Amsterdam and The Scripps Research Institute HIV Vaccine Research and Design Program for their contributions to the design and validation of near-native mimicry for soluble BG505 SOSIP.664 trimers. We thank J. Baalwa, D. Ellenberger, F. Gao, B. Hahn, K. Hong, J. Kim, F. McCutchan, D. Montefiori, L. Morris, J. Overbaugh, E. Sanders-Buell, G. Shaw, R. Swanstrom, M. Thomson, S. Tovanabutra, C. Williamson and L. Zhang for contributing the HIV-1–Env plasmids used in our neutralization panel. Support for this work was provided by the Intramural Research Program of the Vaccine Research Center, National Institute of Allergy and Infectious Diseases (NIAID), US National Institutes of Health (NIH) (to J.A., A.B.M., J.R.M. and P.D.K.); the Division of AIDS, NIAID, NIH (P01-AI100151 to S.Z.-P., P01-AI104722 to L.S., R01-AI93278 to J.M.B., R21-AI100696 to W.M. and S.C.B., R21-AI112389 to K.K.L. and R33-AI84714 to J.M.B.); the US National Institutes of General Medical Sciences (P01-GM56550 to E.F., S.C.B. and W.M., R01-GM78031 to B.R.D. and R01-GM98859 to S.C.B.); the US National Institute of Heart, Lung and Blood (PO1-HL59725 to S.Z.-P.); the US National Science Foundation (MCB-1157506 to E.F.); the Bill and Melinda Gates Foundation Collaboration for AIDS Vaccine Discovery (OPP1033102 to K.K.L.); the Australian Research Council (DP130102219 to L.K.L.); the Irvington Fellows Program of the Cancer Research Program (to J.B.M.); the Department of Veterans Affairs (to S.Z.-P.); and the China Scholarship Council–Yale World Scholars (fellowship to X.M.). This project was funded in part with Federal funds to U.B. from the Frederick National Laboratory for Cancer Research, NIH, under contract HHSN261200800001E. Use of sector 22 (Southeast Region Collaborative Access team) at the Advanced Photon Source was supported by the US Department of Energy, Basic Energy Sciences, Office of Science, under contract no. W-31-109-Eng-38.

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Author notes

  1. Young Do Kwon, Marie Pancera, Priyamvada Acharya and Ivelin S Georgiev: These authors contributed equally to this work.

Authors and Affiliations

  1. Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Young Do Kwon, Marie Pancera, Priyamvada Acharya, Ivelin S Georgiev, Jason Gorman, M Gordon Joyce, Sandeep Narpala, Cinque Soto, Yongping Yang, Tongqing Zhou, Robert T Bailer, Michael Chambers, Gwo-Yu Chuang, Nicole A Doria-Rose, Aliaksandr Druz, Mark A Hallen, Tatsiana Kirys, Mark K Louder, Sijy O'Dell, Gilad Ofek, Madhu Prabhakaran, Mallika Sastry, Guillaume B E Stewart-Jones, Jonathan Stuckey, Paul V Thomas, Tishina Tittley, Baoshan Zhang, Lawrence Shapiro, Adrian B McDermott, John R Mascola & Peter D Kwong

  2. San Diego Biomedical Research Institute, San Diego, California, USA

    Emma T Crooks, Keiko Osawa & James M Binley

  3. Department of Medicinal Chemistry, University of Washington, Seattle, Washington, USA

    Miklos Guttman & Kelly K Lee

  4. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, Connecticut, USA

    Xiaochu Ma, James B Munro & Walther Mothes

  5. Department of Physiology and Biophysics, Weill Cornell Medical College of Cornell University, New York, New York, USA

    Daniel S Terry, Hong Zhao, Zhou Zhou & Scott C Blanchard

  6. Department of Biochemistry & Molecular Biophysics, Columbia University, New York, New York, USA

    Goran Ahlsen & Lawrence Shapiro

  7. Department of Systems Biology, Columbia University, New York, New York, USA

    Goran Ahlsen

  8. Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA

    Mark A Hallen & Bruce R Donald

  9. Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA

    Adam Harned & Ulrich Baxa

  10. New York University School of Medicine, New York, New York, USA

    Constance Williams & Susan Zolla-Pazner

  11. Department of Chemistry, Duke University, Durham, North Carolina, USA

    Bruce R Donald

  12. Department of Computer Science, Duke University, Durham, North Carolina, USA

    Bruce R Donald

  13. Structural and Computational Biology Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia

    Lawrence K Lee

  14. New York Veterans Affairs Harbor Healthcare System, New York, New York, USA

    Susan Zolla-Pazner

  15. Department of Biology, Johns Hopkins University, Baltimore, Maryland, USA

    Arne Schön & Ernesto Freire

  16. Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    James Arthos

  17. Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts, USA

    James B Munro

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  1. Young Do Kwon
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Contributions

Y.D.K. headed the determination of the ligand-free trimer structure; M. Pancera coheaded the conformational fixation and led atomic-level investigations; P.A. coheaded the conformational fixation and led antigenic assessments; and I.S.G. headed the structural compatibility bioinformatics and designed the DS mutation. M. Pancera, T.Z., A.D. and P.D.K. contributed to structure determination; Y.D.K. and C.S. performed structural analysis; R.T.B. and M.K.L. assessed neutralization breadth; I.S.G., G.-Y.C., M.A.H., T.K., B.R.D. and L.K.L. performed structural-compatibility bioinformatics; M. Pancera, P.A., M.G.J., S.N., M.C., G.O., M. Prabhakaran, M.S., T.T., C.W., S.Z.-P. and A.B.M. performed antigenic analyses; J.G., G.B.E.S.-J., Y.Y., B.Z. and J.R.M. contributed to conformational fixation; A.H. and U.B. performed EM; M. Pancera, P.A., A.S. and E.F. performed calorimetry; Y.D.K., G.A. and L.S. performed ultracentrifugation; Y.D.K., M.G. and K.K.L. performed and analyzed HDX-MS; N.A.D.-R., S.O. and J.R.M. created and analyzed mutant virus; J.G., X.M., D.S.T., H.Z., Z.Z., J.A., J.B.M., S.C.B. and W.M. performed smFRET; P.A., M.G.J. and P.V.T. assessed physical and temporal stability; M. Pancera, E.T.C., K.O. and J.M.B. contributed VLP analysis; and I.S.G., J.S. and P.D.K. evaluated information flow. Y.D.K., M. Pancera, P.A., I.S.G. and P.D.K. assembled and wrote the paper, on which all principal investigators commented.

Corresponding author

Correspondence to Peter D Kwong.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Residue-level properties of mature ligand-free HIV-1 BG505 SOSIP.664 Env.

(a) One of the three gp120 protomers is shown in red with residue numbers of N/C termini. gp41 is shown in rainbow colors. Glycans are shown in magenta. (b) V1V2 and V3 domains of gp120 are shown in orange and blue, respectively. gp41 is shown in pink. (c) After superposition of 98 ligand-free or antibody-bound gp120 structures, the average Cα-movement was computed. The gp120 is shown with regions of less (or greater) than 2 Å movement in green (or magenta). (d) gp41 is shown with the same color code as in c. (e) The color scale ranges from grey to purple for the conserved residues to the variable, respectively. (f) Polar residues (Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, and Thr) are shown in blue. Hydrophobic residues (Ala, Cys, Phe, Ile, Leu, Met, Pro, Val, Trp, and Tyr) are colored in yellow. (g) Real-space correlation coefficient (CC) of each residue is shown in rainbow colors (blue, CC greater than 0.9, to red, CC less than 0.6). (h) The color scale ranges from blue to red for H-D exchange of 0 to 75% in 3 seconds, respectively. Segments where no H-D exchange rate is available are colored in grey.

Supplementary Figure 2 Surface-level properties of mature ligand-free HIV-1 BG505 SOSIP.664 Env.

(a) Three gp120-gp41 protomers are colored pink, green, and wheat in surface representation. (b) V1V2 and V3 domain of gp120 are shown in orange and blue, respectively. gp41 is shown in pink. (c) After superposition of 98 ligand-free or antibody-bound gp120 structures, the average Cα-movement was computed. The gp120 is shown with regions of less (or greater) than 2 Å movement in green (or magenta). (d) gp41 is shown with the same color code as in c. (e) The color scale ranges from white to purple from conserved to variable residues, respectively. (f) Polar residues (Asp, Glu, His, Lys, Asn, Gln, Arg, Ser, and Thr) are shown in blue. Hydrophobic residues (Ala, Cys, Phe, Ile, Leu, Met, Pro, Val, Trp, and Tyr) are colored in yellow. (g) Electrostatic potentials. (h) The color scale ranges from blue to red for H-D exchange of 0 to 75% in 3 seconds, respectively. Segments where no H-D exchange rate is available are colored in grey.

Supplementary Figure 3 B factors of prefusion ligand-free trimer versus prefusion-to-postfusion movement.

Left panel. Pre-fusion ligand-free trimers of the type 1 fusion glycoproteins of HIV-1, influenza A virus (Influenza) and respiratory syncytial virus (RSV) are shown in ribbon representation with one protomer colored by B-factors (scale shown under each trimers). Middle panel. Same pre-fusion ligand-free trimers are shown with one protomer colored by Cα movement between pre- and post-fusion conformations (with scale shown). Right panel. Relationship between pre-fusion Env B-factor and Cα-subunit movement between pre- and post-fusion conformations.

Supplementary Figure 4 Glycosylation of the endoglycosidase H–treated ligand-free BG505 SOSIP.664 trimer, its crystal packing and comparison of real-space correlation between 35O22- and PGT122-bound and ligand-free BG505 SOSIP.664.

(a) A gp120-gp41 protomer of the ligand-free BG505 SOSIP.664 trimer is shown in ribbon representation (gp120 in red and gp41 in rainbow colors). Another protomer is shown in ribbon representation (grey) with glycans in stick representation (magenta). For clarity, one protomer is not shown. All N-linked glycans were reduced to a single proximal N-acetyl glucosamine, except for glycans at residues 197, 262, and 332, where additional monosaccharide residues were observed, or at residue 137, which was mostly disordered. (b) P63 crystal packing of the ligand-free trimer. Regions where crystal contacts may occur or two inter molecules are in close proximity are highlighted. These areas include i) Helix α9 (residues 649-662) and residues 458-462, ii) Helix α0 (residues 60-73) and Helix α8 (residues 618-626), iii) residues 232-234 and residues 323-325, and iv) residues 134-137 and 347-352. (c) A protomer of each trimer is shown in a color gradient to highlight regions of model uncertainty according to its real-space correlation coefficient.

Supplementary Figure 5 Structural compatibility of ligand-free trimer with broadly neutralizing and ineffective antibodies.

(a-b) Ligand-free Env structure is displayed as a Cα-ribbon mapping per-residue RMSD a, and antibody-antigen volume overlap b, for broadly neutralizing (left) or ineffective (right) antibodies. (a) Each residue that is part of an antibody epitope is colored green if the RMSD between the ligand-free and antibody-bound conformation for the given residue is less than 2 Å and magenta if greater than 2 Å; residues that are part of multiple antibody epitopes are colored according to the highest RMSD value associated with them. (b) All epitope residues for a given antibody are colored green if the antibody-antigen volume overlap is less than 500 Å3 for the antibody and magenta if greater than 500 Å3; residues that are part of multiple epitopes are colored green if the volume overlap for at least one of these epitopes is less than 500 Å3 and magenta otherwise. Non-epitope regions are shown in grey. (c) Examples of antibody-antigen volume overlap. Shown are antibodies F105 (left) and 447-52D (right) in surface representation, with visible overlap upon epitope alignment to the ligand-free trimer (in white/grey cartoon and transparent surface representation). Antibody segments that include regions of overlap are highlighted in red.

Supplementary Figure 6 Characterization of purified BG505 SOSIP.664 and selected variants.

(a) Properties of purified gp140 proteins. *the percentage of trimers was obtained by measuring area under the curve of the gel filtration profile of the various peaks representing aggregates, gp140 trimer, dimer and monomer. (b) Gel filtration profiles on Superdex 200 with blue line showing a second round of purification when performed. Dotted lines show the fractions selected for analyses. (c) 2D class averages from a reference-free classification of negative stained EM data for each protein. Box size = 28 nm.

Supplementary Figure 7 HDX of BG505 SOSIP.664 201C 433C variant (DS-SOSIP.664) and BG505 SOSIP.664 (WT).

(a) HDX comparison of BG505 SOSIP.664 201C-433C variant (DS-SOSIP.664) and BG505 SOSIP.664 (WT). Butterfly plots show the exchange profile of ligand-free SOSIP.664 (positive axis) and DS-SOSIP.664 (negative axis). The percent exchange at 3 sec (orange), 1 min (red), 30 min (blue) and 20 hr (black) is shown for each observable peptide at the midpoint of its position in the primary sequence. The difference plots below show the raw difference at each time point between the two data sets. Only very minor differences are observed between the HDX profiles of WT and DS-SOSIP.664. (b, c) Butterfly plots showing the changes upon CD4 binding to DS-SOSIP (b) or WT SOSIP.664 (c). The difference plots reveal regions becoming less protected upon CD4 binding (below zero) and regions becoming more protected (above zero) with large changes highlighted and labeled. Individual exchange plots for all observable peptides with error bars are shown in Supplementary Data Set 6.

Supplementary Figure 8 Immune evasion by type 1 fusion glycoproteins and the effect of DS-SOSIP on HIV-1 Env.

Mechanisms of immune evasion (glycan shield, genetic variation and conformational masking) for the type 1 fusion glycoproteins of HIV-1, influenza A virus (Influenza) and respiratory syncytial virus (RSV) are depicted in surface representation, with N-linked glycosylation (green, top panels) and sequence variation (purple gradient, middle panel) highlighted as previously shown in Pancera et al, Nature, 2014. Influenza virus hemagglutinin remains in its pre-fusion state if not exposed to acidic pH, while the RSV fusion glycoprotein readily transitions from its pre-fusion conformation. While the pre-fusion conformation of HIV-1 Env is metastable, SOSIP.664 alterations allow for a stable pre-fusion closed conformation; the 201C-433C ‘DS” mutation additionally stabilizes HIV-1 Env so that it is no longer triggered by the CD4 receptor to expose ineffective epitopes.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Tables 1–7 and Supplementary Note (PDF 4677 kb)

Supplementary Data Sets 1–6

Supplementary Data Sets 1–6 (PDF 2654 kb)

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Do Kwon, Y., Pancera, M., Acharya, P. et al. Crystal structure, conformational fixation and entry-related interactions of mature ligand-free HIV-1 Env. Nat Struct Mol Biol 22, 522–531 (2015). https://doi.org/10.1038/nsmb.3051

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