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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Starcich, B.R. et al. Identification and characterization of conserved and variable regions in the envelope gene of HTLV-III/LAV, the retrovirus of AIDS. Cell 45, 637–648 (1986).
Wei, X. et al. Antibody neutralization and escape by HIV-1. Nature 422, 307–312 (2003).
Chen, L. et al. Structural basis of immune evasion at the site of CD4 attachment on HIV-1 gp120. Science 326, 1123–1127 (2009).
Kwong, P.D. et al. HIV-1 evades antibody-mediated neutralization through conformational masking of receptor-binding sites. Nature 420, 678–682 (2002).
Fouts, T.R., Binley, J.M., Trkola, A., Robinson, J.E. & Moore, J.P. Neutralization of the human immunodeficiency virus type 1 primary isolate JR-FL by human monoclonal antibodies correlates with antibody binding to the oligomeric form of the envelope glycoprotein complex. J. Virol. 71, 2779–2785 (1997).
Huang, C.C. et al. Structure of a V3-containing HIV-1 gp120 core. Science 310, 1025–1028 (2005).
Weiss, R.A. et al. Neutralization of human T-lymphotropic virus type III by sera of AIDS and AIDS-risk patients. Nature 316, 69–72 (1985).
Bures, R. et al. Immunization with recombinant canarypox vectors expressing membrane- anchored glycoprotein 120 followed by glycoprotein 160 boosting fails to generate antibodies that neutralize R5 primary isolates of human immunodeficiency virus type 1. AIDS Res. Hum. Retroviruses 16, 2019–2035 (2000).
Flynn, N.M. et al. Placebo-controlled phase 3 trial of a recombinant glycoprotein 120 vaccine to prevent HIV-1 infection. J. Infect. Dis. 191, 654–665 (2005).
Liu, J., Bartesaghi, A., Borgnia, M.J., Sapiro, G. & Subramaniam, S. Molecular architecture of native HIV-1 gp120 trimers. Nature 455, 109–113 (2008).
Bartesaghi, A., Merk, A., Borgnia, M.J., Milne, J.L. & Subramaniam, S. Prefusion structure of trimeric HIV-1 envelope glycoprotein determined by cryo-electron microscopy. Nat. Struct. Mol. Biol. 20, 1352–1357 (2013).
Julien, J.P. et al. Crystal structure of a soluble cleaved HIV-1 envelope trimer. Science 342, 1477–1483 (2013).
Lyumkis, D. et al. Cryo-EM structure of a fully glycosylated soluble cleaved HIV-1 envelope trimer. Science 342, 1484–1490 (2013).
Pancera, M. et al. Structure and immune recognition of trimeric pre-fusion HIV-1 Env. Nature 514, 455–461 (2014).
Wu, X. et al. Neutralization escape variants of human immunodeficiency virus type 1 are transmitted from mother to infant. J. Virol. 80, 835–844 (2006).
Binley, J.M. et al. A recombinant human immunodeficiency virus type 1 envelope glycoprotein complex stabilized by an intermolecular disulfide bond between the gp120 and gp41 subunits is an antigenic mimic of the trimeric virion-associated structure. J. Virol. 74, 627–643 (2000).
Sanders, R.W. et al. Stabilization of the soluble, cleaved, trimeric form of the envelope glycoprotein complex of human immunodeficiency virus type 1. J. Virol. 76, 8875–8889 (2002).
Sanders, R.W. et al. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies. PLoS Pathog. 9, e1003618 (2013).
Kwon, Y.D. et al. Unliganded HIV-1 gp120 core structures assume the CD4-bound conformation with regulation by quaternary interactions and variable loops. Proc. Natl. Acad. Sci. USA 109, 5663–5668 (2012).
Kwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648–659 (1998).
Zhou, T. et al. Structural definition of a conserved neutralization epitope on HIV-1 gp120. Nature 445, 732–737 (2007).
Zhou, T. et al. Structural basis for broad and potent neutralization of HIV-1 by antibody VRC01. Science 329, 811–817 (2010).
Pejchal, R. et al. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield. Science 334, 1097–1103 (2011).
Scharf, L. et al. Antibody 8ANC195 reveals a site of broad vulnerability on the HIV-1 envelope spike. Cell Reports 7, 785–795 (2014).
Wyatt, R. & Sodroski, J. The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens. Science 280, 1884–1888 (1998).
Chan, D.C., Fass, D., Berger, J.M. & Kim, P.S. Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263–273 (1997).
Weissenhorn, W., Dessen, A., Harrison, S.C., Skehel, J.J. & Wiley, D.C. Atomic structure of the ectodomain from HIV-1 gp41. Nature 387, 426–430 (1997).
Munro, J.B. et al. Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions. Science 346, 759–763 (2014).
Guttman, M. et al. Antibody potency relates to the ability to recognize the closed, pre-fusion form of HIV Env. Nat. Commun. 6, 6144 (2015).
Jancarik, J.K.S.-H. Sparse matrix sampling: a screening method for crystallization of proteins. J. Appl. Crystallogr. 24, 409–411 (1991).
Majeed, S. et al. Enhancing protein crystallization through precipitant synergy. Structure 11, 1061–1070 (2003).
Adams, P.D. et al. Recent developments in the PHENIX software for automated crystallographic structure determination. J. Synchrotron Radiat. 11, 53–55 (2004).
Korber, B.T., Foley, B.T., Kuiken, C.L., Pillai, S.K. & Sodroski, J.G. in Human Retroviruses and AIDS 1998: a Compilation and Analysis of Nucleic Acid and Amino Acid Sequences Part III (eds. Korber, B., Kuiken, C.L., Foley, B., Hahn, B., McCutchan, F., Mellors, J.W. & Sodroski, J.) 102–111 (Los Alamos National Laboratory, 1998).
Burton, D.R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 1024–1027 (1994).
Huang, J. et al. Broad and potent neutralization of HIV-1 by a human antibody that binds the gp41–gp120 interface. Nature 515, 138–142 (2014).
Walker, L.M. et al. Broad neutralization coverage of HIV by multiple highly potent antibodies. Nature 477, 466–470 (2011).
Zolla-Pazner, S. et al. The cross-clade neutralizing activity of a human monoclonal antibody is determined by the GPGR V3 motif of HIV type 1. AIDS Res. Hum. Retroviruses 20, 1254–1258 (2004).
Liao, H.-X. et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469–476 (2013).
Guenaga, J. et al. Well-ordered trimeric HIV-1 subtype B and C soluble spike mimetics generated by negative selection display native-like properties. PLoS Pathog. 11, e1004570 (2015).
Mbah, H.A. et al. Effect of soluble CD4 on exposure of epitopes on primary, intact, native human immunodeficiency virus type 1 virions of different genetic clades. J. Virol. 75, 7785–7788 (2001).
Stanfield, R.L., Gorny, M.K., Williams, C., Zolla-Pazner, S. & Wilson, I.A. Structural rationale for the broad neutralization of HIV-1 by human monoclonal antibody 447–52D. Structure 12, 193–204 (2004).
Jiang, X. et al. Conserved structural elements in the V3 crown of HIV-1 gp120. Nat. Struct. Mol. Biol. 17, 955–961 (2010).
Thali, M. et al. Characterization of conserved human immunodeficiency virus type 1 gp120 neutralization epitopes exposed upon gp120–CD4 binding. J. Virol. 67, 3978–3988 (1993).
McLellan, J.S. et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus. Science 342, 592–598 (2013).
Wu, X. et al. Rational design of envelope identifies broadly neutralizing human monoclonal antibodies to HIV-1. Science 329, 856–861 (2010).
Doria-Rose, N.A. et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55–62 (2014).
Posner, M.R., Cavacini, L.A., Emes, C.L., Power, J. & Byrn, R. Neutralization of HIV-1 by F105, a human monoclonal antibody to the CD4 binding site of gp120. J. Acquir. Immune Defic. Syndr. 6, 7–14 (1993).
Arthos, J. et al. Biochemical and biological characterization of a dodecameric CD4-Ig fusion protein: implications for therapeutic and vaccine strategies. J. Biol. Chem. 277, 11456–11464 (2002).
Daar, E.S. & Ho, D.D. Relative resistance of primary HIV-1 isolates to neutralization by soluble CD4. Am. J. Med. 90, 22S–26S (1991).
Crooks, E.T., Tong, T., Osawa, K. & Binley, J.M. Enzyme digests eliminate nonfunctional Env from HIV-1 particle surfaces, leaving native Env trimers intact and viral infectivity unaffected. J. Virol. 85, 5825–5839 (2011).
Tong, T., Crooks, E.T., Osawa, K. & Binley, J.M. HIV-1 virus-like particles bearing pure env trimers expose neutralizing epitopes but occlude nonneutralizing epitopes. J. Virol. 86, 3574–3587 (2012).
Trkola, A. et al. Human monoclonal antibody 2G12 defines a distinctive neutralization epitope on the gp120 glycoprotein of human immunodeficiency virus type 1. J. Virol. 70, 1100–1108 (1996).
McLellan, J.S. et al. Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody. Science 340, 1113–1117 (2013).
McLellan, J.S., Yang, Y., Graham, B.S. & Kwong, P.D. Structure of respiratory syncytial virus fusion glycoprotein in the postfusion conformation reveals preservation of neutralizing epitopes. J. Virol. 85, 7788–7796 (2011).
Swanson, K.A. et al. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers. Proc. Natl. Acad. Sci. USA 108, 9619–9624 (2011).
Kovacs, J.M. et al. Stable, uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure. Proc. Natl. Acad. Sci. USA 111, 18542–18547 (2014).
Pancera, M. et al. Soluble mimetics of human immunodeficiency virus type 1 viral spikes produced by replacement of the native trimerization domain with a heterologous trimerization motif: characterization and ligand binding analysis. J. Virol. 79, 9954–9969 (2005).
Sanders, R.W. et al. HIV-1 neutralizing antibodies induced by native-like envelope trimers. Science http://dx.doi.org/10.1126/science.aac4223 (18 June 2015).
McLellan, J.S. et al. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9. Nature 480, 336–343 (2011).
Tran, E.E. et al. Structural mechanism of trimeric HIV-1 envelope glycoprotein activation. PLoS Pathog. 8, e1002797 (2012).
Killikelly, A. et al. Thermodynamic signatures of the antigen binding site of mAb 447–52D targeting the third variable region of HIV-1 gp120. Biochemistry 52, 6249–6257 (2013).
Stanfield, R.L., Gorny, M.K., Zolla-Pazner, S. & Wilson, I.A. Crystal structures of human immunodeficiency virus type 1 (HIV-1) neutralizing antibody 2219 in complex with three different V3 peptides reveal a new binding mode for HIV-1 cross-reactivity. J. Virol. 80, 6093–6105 (2006).
Gorny, M.K. et al. Human anti-V3 HIV-1 monoclonal antibodies encoded by the VH5–51/VL lambda genes define a conserved antigenic structure. PLoS ONE 6, e27780 (2011).
Rini, J.M. et al. Crystal structure of a human immunodeficiency virus type 1 neutralizing antibody, 50.1, in complex with its V3 loop peptide antigen. Proc. Natl. Acad. Sci. USA 90, 6325–6329 (1993).
Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).
Bernstein, F.C. et al. The Protein Data Bank: a computer-based archival file for macromolecular structures. J. Mol. Biol. 112, 535–542 (1977).
Zhang, Y. & Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res. 33, 2302–2309 (2005).
Pancera, M. et al. Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility. Proc. Natl. Acad. Sci. USA 107, 1166–1171 (2010).
Guttman, M. et al. CD4-induced activation in a soluble HIV-1 Env trimer. Structure 22, 974–984 (2014).
Sanner, M.F., Olson, A.J. & Spehner, J.C. Reduced surface: an efficient way to compute molecular surfaces. Biopolymers 38, 305–320 (1996).
Nicholls, A., Sharp, K.A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991).
Sarzotti-Kelsoe, M. et al. Optimization and validation of the TZM-bl assay for standardized assessments of neutralizing antibodies against HIV-1. J. Immunol. Methods 409, 131–146 (2014).
Huang, C.C. et al. Structural basis of tyrosine sulfation and VH-gene usage in antibodies that recognize the HIV type 1 coreceptor-binding site on gp120. Proc. Natl. Acad. Sci. USA 101, 2706–2711 (2004).
Xiang, S.H. et al. Epitope mapping and characterization of a novel CD4-induced human monoclonal antibody capable of neutralizing primary HIV-1 strains. Virology 315, 124–134 (2003).
Huang, J. et al. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface. Nature 515, 138–142 (2014).
Scheid, J.F. et al. Broad diversity of neutralizing antibodies isolated from memory B cells in HIV-infected individuals. Nature 458, 636–640 (2009).
Liao, H.X. et al. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus. Nature 496, 469–476 (2013).
Calarese, D.A. et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071 (2003).
Georgiev, I.S. et al. Delineating antibody recognition in polyclonal sera from patterns of HIV-1 isolate neutralization. Science 340, 751–756 (2013).
Doria-Rose, N.A. et al. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies. Nature 509, 55–62 (2014).
Seaman, M.S. et al. Tiered categorization of a diverse panel of HIV-1 Env pseudoviruses for assessment of neutralizing antibodies. J. Virol. 84, 1439–1452 (2010).
Dreyfus, C. et al. Highly conserved protective epitopes on influenza B viruses. Science 337, 1343–1348 (2012).
Tharakaraman, K., Subramanian, V., Cain, D., Sasisekharan, V. & Sasisekharan, R. Broadly neutralizing influenza hemagglutinin stem-specific antibody CR8020 targets residues that are prone to escape due to host selection pressure. Cell Host Microbe 15, 644–651 (2014).
Palivizumab, a humanized respiratory syncytial virus monoclonal antibody, reduces hospitalization from respiratory syncytial virus infection in high-risk infants. The IMpact-RSV Study Group. Pediatrics 102, 531–537 (1998).
Mastronarde, D.N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).
Shu, Y. et al. Efficient protein boosting after plasmid DNA or recombinant adenovirus immunization with HIV-1 vaccine constructs. Vaccine 25, 1398–1408 (2007).
Wei, X. et al. Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapy. Antimicrob. Agents Chemother. 46, 1896–1905 (2002).
Tong, T., Osawa, K., Robinson, J.E., Crooks, E.T. & Binley, J.M. Topological analysis of HIV-1 glycoproteins expressed in situ on virus surfaces reveals tighter packing but greater conformational flexibility than for soluble gp120. J. Virol. 87, 9233–9249 (2013).
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.
The authors declare no competing financial interests.
Integrated supplementary information
(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.
(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.
(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.
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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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