Structural characterization of a highly-potent V3-glycan broadly neutralizing antibody bound to natively-glycosylated HIV-1 envelope

Abstract

Broadly neutralizing antibodies (bNAbs) isolated from HIV-1-infected individuals inform HIV-1 vaccine design efforts. Developing bNAbs with increased efficacy requires understanding how antibodies interact with the native oligomannose and complex-type N-glycan shield that hides most protein epitopes on HIV-1 envelope (Env). Here we present crystal structures, including a 3.8-Å X-ray free electron laser dataset, of natively glycosylated Env trimers complexed with BG18, the most potent V3/N332_gp120 glycan-targeting bNAb reported to date. Our structures show conserved contacts mediated by common D gene-encoded residues with the N332_gp120 glycan and the gp120 GDIR peptide motif, but a distinct Env-binding orientation relative to PGT121/10-1074 bNAbs. BG18's binding orientation provides additional contacts with N392_gp120 and N386_gp120 glycans near the V3-loop base and engages protein components of the V1-loop. The BG18-natively-glycosylated Env structures facilitate understanding of bNAb-glycan interactions critical for using V3/N332_gp120 bNAbs therapeutically and targeting their epitope for immunogen design.

Fig. 1

Crystal structures of natively glycosylated HIV-1 Env trimers complexed with BG18. a Cartoon representation of the 3.8 Å structure of BG505 Env (gp120, light gray; gp41, dark gray) in complex with BG18 (blue) and 35O22 (orange) Fabs. Ordered, native high-mannose glycans (cyan) are represented as sticks, and complex-type glycans (salmon) are shown as spheres. b 4.9 Å structure of B41 Env bound to BG18 (blue) and 35O22 (orange). Glycans shown as cyan spheres. c 6.7 Å structure of BG505 Env bound to BG18 (blue) and IOMA (green) Fabs. Glycans were not modeled due to the limited resolution. d Close-up of the BG18 binding site (V_H in dark blue, V_L in light blue superimposed on the final 2F_o−F_calc electron density map contoured at 1.2σ) on the surface of gp120 (gray) from the BG18-BG505-35O22 structure. Ordered glycans (cyan) near the BG18 binding site are represented as sticks

Fig. 2

Comparison of apo- and Env-bound BG18 variable domains. a Superposition of V_H-V_L domains (230 Cα atoms) of unliganded BG18 (deep teal; PDB 5UD9 (http://dx.doi.org10.2210/pdb5UD9/pdb)) with BG18 in the BG18-BG505 structure (blue) showed conservation of the BG18 antigen-binding site and ordering of CDRL2 in the BG505-bound structure (dashed red line represents disordered CDRL2 in unliganded BG18). b Surface representation of BG18 in the BG505-bound structure showed conservation of BG18 clefts, including the cleft between CDRH3 and CDRL1/3 loops observed in the BG18 apo structure. c Ribbon and cartoon representation of the overlay between BG18 (aligned on the Fab V_H domains) in the BG18-BG505-35O22 complex (blue), unliganded BG18 (deep teal), and unliganded 10-1074 (magenta; PDB 4FQ2 (http://dx.doi.org10.2210/pdb4FQ2/pdb)). CDRH3 loops for the three structures are represented as cartoons. Inset: overlay of BG18-BG505 and 10-1074-BG505 (PDB 5T3Z (http://dx.doi.org10.2210/pdb5T3Z/pdb)) CDRH3 loops. d Cartoon and stick representation of BG18 CDRH3 recognition of the N332_gp120 glycan. BG18 residues comprising a conserved structural motif (R-I-Y-G-V/I-I) are labeled. Electron density contoured at 1σ from 2F_obs−F_calc composite annealed omit map calculated with phases from models with the N332_gp120 glycan and BG18 CDRH3 coordinates omitted to reduce potential phase bias (cyan and blue mesh)

Fig. 3

Distinct orientation on HIV-1 Env of BG18 compared to other V3/N332_gp120 bNAbs. BG18 Fab (blue, surface) orientation on gp120 (gray, surface) overlaid with 10-1074 (a, magenta; PDB 5T3Z (http://dx.doi.org10.2210/pdb5T3Z/pdb)) and PGT122 (b, yellow; 5FYL (http://dx.doi.org10.2210/pdb5T3Z/pdb)) and PGT122 (b, yellow; 5FYL (http://dx.doi.org10.2210/pdb5FYL/pdb)) Fabs. c Overlay of CDRH3 loop of BG18 (blue), 10-1074 (magenta), PGT122 (yellow), PGT121 precursor (red; PDB 5CEZ (http://dx.doi.org10.2210/pdb5CEZ/pdb)), and PGT124 (orange; PDB 4R2G (http://dx.doi.org10.2210/pdb4R2G/pdb)) after alignment of bound gp120s (gray, surface) demonstrating a conserved structural motif recognizing the N332_gp120 glycan (cyan). d, e Comparison of V_H-V_L domains of PGT122 (yellow) and 10-1074 (magenta) with BG18 (blue). The V_H-V_L domain orientation of BG18 on Env trimer is related by a 78˚ rotation and 6.8 Å translation about the indicated axis (black dashed arrow) to the 10-1074 variable domains after alignment against gp120 in the BG18-Env and 10-1074-Env structures

Fig. 4

Glycan interactions with V3/N332_gp120 bNAbs in structures including natively glycosylated Env. a, b Comparison of the orientations on gp120 (gray surface and cartoon) of the CDR loops from (a) BG18 (blue, ribbon) and (b) 10-1074 (magenta, ribbon) demonstrating that the BG18 variable domains are rotated clockwise about CDRH3 relative to the 10-1074 variable domains. The distinct BG18 orientation on gp120 resulted in contacts with N156_gp120, N386_gp120, and N392_gp120 glycans (cyan, sticks) in proximity to the V3-base. Red dotted outline: Differences in the N332_gp120 glycan conformation on BG505 Env bound to BG18 (a) or to 10-1074 (b). c Close-up of BG18 interaction with the N332_gp120 glycan showing CDRH3, CDRH1 and CDRL2 loops at the glycan interface. d Overlay of BG18-BG505 and 10-1074–BG505 (PDB 5T3Z (http://dx.doi.org10.2210/pdb5T3Z/pdb)) structures showed that the BG18-bound N332_gp120 glycan conformation would clash (yellow star) with light chain CDR loops of 10-1074 and other PGT121-like bNAbs that display nearly identical binding modes. e Surface representation of BG18 interactions with the N392_gp120 and N386_gp120 glycans showing the N392_gp120 glycan buried (~800 Ų total BSA) inside cleft 2 located between the CDRH3 and CDRL1/3 loops. The N386_gp120 glycan associates weakly with light chain BG18 (45 Ų total BSA), but forms branch–branch interactions with the N392_gp120 glycan. c, e Electron density contoured at 1σ from 2F_obs−F_calc composite annealed omit maps calculated with phases from models with glycan coordinates omitted to reduce potential phase bias (gray mesh)

Fig. 5

Comparison of GDIR recognition by BG18 and 10-1074. a Cartoon and stick representation of BG18 CDRH3 (dark blue), BG18 CDL2 (light blue), and gp120 (gray; residues 318-330). Electron density contoured at 1σ from 2F_obs−F_calc final refined electron density map with −100 Ų B-sharpening is shown (gray mesh). b BG18 interactions with the gp120 GDIR motif (G324g_p120-D325g_p120-I326g_p120-R327g_p120) at the base of the V3-loop. In common with the PGT121-like bNAbs, CDRH3 E111_HC forms a potential salt bridge with R327_gp120, while CDRH3 Y104_HC stacks against D325_gp120. In addition, CDRL2 residues S53_LC and Q54_LC engage D325_gp120 forming potential H-bond interactions. c Comparison of BG18 (light blue) and 10-1074 (pink; PDB 5T3Z (http://dx.doi.org10.2210/pdb5T3Z/pdb)) interactions with D325_gp120 in the GDIR motif. Engagement of the carboxylate group of D325_gp120 is achieved by residues in CDRL2 (BG18) or serine residues in CDRs L1 and L3 (10-1074). Potential H-bonds represented as dashed lines. d Alignment of sequences of inferred germline and mature light chains of BG18 and 10-1074. The LV3-21*02 germline V gene segment is used for both 10-1074 and PGT121-like antibodies. Red asterisks indicate residues involved in D325_gp120 recognition in the GDIR motif as shown in c

Fig. 6

BG18 contacts with gp120 V1-loop. a Surface and cartoon representation of BG18 V_H (dark blue) and V_L (light blue) loops involved in gp120 V1-loop (gray; residues 128–158, disordered residues depicted as dashed red line) interactions. Electron density contoured at 1σ from a 2F_obs−F_calc composite annealed omit map is shown (gray mesh). Glycans at position N133_gp120 and N156_gp120 are shown. b Cartoon and stick representation of residue interactions between BG18 V_L domain (light blue) and the gp120 V1-loop (gray). Potential H-bonding occurs between T139_gp120 and BG18 T30_LC in the CDRL1 loop. In addition, BG18 W67_LC in FWRL3 stacks against I138_gp120. BG18 contacts with gp120 positions 138 and 139 are likely specific to Envs with V1 characteristics similar to BG505, since similar conformations were not observed in our BG18-B41 structure. H-bonds and pi-stacking are indicated by black dashed lines. Red asterisk on N137_gp120 indicates a PNGS. c Electrostatic surface potentials with red indicating negative electrostatic potential and blue indicating positive electrostatic potential for BG18 shown with cartoon and stick representation of nearby gp120 elements. BG18 includes a positively charged cleft in the vicinity of the gp120 V1-loop (dashed line indicates disordered region), which may provide increased protein–protein interactions with the gp120 surface in HIV-1 strains harboring charged residues in the V1-loop