Figure 4.
Structural analyses of the BG505 SOSIP.664-7-269 IgA Fab complex. (A) Side view (left) and top view (right) of the 2.8-Å single-particle cryo-EM reconstruction of the BG505 SOSIP.664-7-269-3BNC117 complex colored by components (dark gray, gp41; light gray, gp120; blue, 7-269 VH; light-blue, 7-269 VL; pink, 3BNC117 Fab; green, N-glycans). (B) Structure of the BG505 SOSIP.664-7-269 complex with other anti-glycan V3 bNAbs superimposed. One protomer was aligned to gp120 in the complex with 10-1074 (PDB accession no. 5T3Z), while another protomer was superimposed to the structure of the Env with 2G12 (PDB accession no. 6OZC). Only the variable heavy and light chains are shown, and the N332 glycan is indicated (green). (C) Structure of the HIV-1 Env-7-269 protomer highlighting in sticks the glycans that establish major (N295 and N332) and minor (N262, N411) contacts with the antibody. The inset in the top right corner presents the glycan residues modeled at each position, indicating with filled symbols those in contact with 7-269. Squares and circles represent N-acetylglucosamine and mannose residues, respectively. (D) Mapping of the 7-269 epitope (colored in blue) on the density from the EM map corresponding to the BG505 SOSIP.664 trimer. The glycans in the interface are shown in sticks, with the sugar residues forming the epitope in blue. (E) Surface representation of the 7-269 variable domains with residues contacting a particular glycan (or glycan and gp120) indicated in the same color. (F) Structural superposition of the IgH and IgL variable domains (VH and VL, respectively) from 7-269 and anti-glycan-V3 bNAbs that have either a protruding CDRH3 with a β-hairpin (left; 10-1074 [PDB accession no. 5T3Z] and 3H+109L [PDB accession no. 5CEZ]) or an extended or short CDRH3 (438-B11 [PDB accession no. 6UUH], BG18 [PDB accession no. 6CH7], and 2G12 [PDB accession no. 6OZC]). (G) Structural superposition of the variable domains from 7-269 and anti-HIV bNAbs harboring an intra-CDRH3 disulfide bond (yellow sticks) and targeting the glycans-V3 (438-B11 [PDB accession no. 6UUH]), glycans-V1/2 (CAP256-VR26.03 [PDB accession no. 4OD1]), and the CD4bs (45-VRC01.H5.F-117225 [PDB accession no. 4S1S]). (H) ELISA graphs comparing the Env binding of 7-269 and associated CDRH3 cysteine mutant antibodies. Means ± SD of duplicate OD405nm values are shown (representative of two independent experiments). (I) Heatmap comparing the neutralizing activity of 7-269 and associated CDRH3 cysteine mutant antibodies as measured in the TZM-bl assay. Representative data of two independent experiments are shown. <, IC50 below the depicted value.

Structural analyses of the BG505 SOSIP.664-7-269 IgA Fab complex. (A) Side view (left) and top view (right) of the 2.8-Å single-particle cryo-EM reconstruction of the BG505 SOSIP.664-7-269-3BNC117 complex colored by components (dark gray, gp41; light gray, gp120; blue, 7-269 VH; light-blue, 7-269 VL; pink, 3BNC117 Fab; green, N-glycans). (B) Structure of the BG505 SOSIP.664-7-269 complex with other anti-glycan V3 bNAbs superimposed. One protomer was aligned to gp120 in the complex with 10-1074 (PDB accession no. 5T3Z), while another protomer was superimposed to the structure of the Env with 2G12 (PDB accession no. 6OZC). Only the variable heavy and light chains are shown, and the N332 glycan is indicated (green). (C) Structure of the HIV-1 Env-7-269 protomer highlighting in sticks the glycans that establish major (N295 and N332) and minor (N262, N411) contacts with the antibody. The inset in the top right corner presents the glycan residues modeled at each position, indicating with filled symbols those in contact with 7-269. Squares and circles represent N-acetylglucosamine and mannose residues, respectively. (D) Mapping of the 7-269 epitope (colored in blue) on the density from the EM map corresponding to the BG505 SOSIP.664 trimer. The glycans in the interface are shown in sticks, with the sugar residues forming the epitope in blue. (E) Surface representation of the 7-269 variable domains with residues contacting a particular glycan (or glycan and gp120) indicated in the same color. (F) Structural superposition of the IgH and IgL variable domains (VH and VL, respectively) from 7-269 and anti-glycan-V3 bNAbs that have either a protruding CDRH3 with a β-hairpin (left; 10-1074 [PDB accession no. 5T3Z] and 3H+109L [PDB accession no. 5CEZ]) or an extended or short CDRH3 (438-B11 [PDB accession no. 6UUH], BG18 [PDB accession no. 6CH7], and 2G12 [PDB accession no. 6OZC]). (G) Structural superposition of the variable domains from 7-269 and anti-HIV bNAbs harboring an intra-CDRH3 disulfide bond (yellow sticks) and targeting the glycans-V3 (438-B11 [PDB accession no. 6UUH]), glycans-V1/2 (CAP256-VR26.03 [PDB accession no. 4OD1]), and the CD4bs (45-VRC01.H5.F-117225 [PDB accession no. 4S1S]). (H) ELISA graphs comparing the Env binding of 7-269 and associated CDRH3 cysteine mutant antibodies. Means ± SD of duplicate OD405nm values are shown (representative of two independent experiments). (I) Heatmap comparing the neutralizing activity of 7-269 and associated CDRH3 cysteine mutant antibodies as measured in the TZM-bl assay. Representative data of two independent experiments are shown. <, IC50 below the depicted value.

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