Dissection of the carbohydrate specificity of the broadly neutralizing anti-HIV-1 antibody 2G12

Abstract

Human antibody 2G12 neutralizes a broad range of HIV-1 isolates. Hence, molecular characterization of its epitope, which corresponds to a conserved cluster of oligomannoses on the viral envelope glycoprotein gp120, is a high priority in HIV vaccine design. A prior crystal structure of 2G12 in complex with Man(9)GlcNAc(2) highlighted the central importance of the D1 arm in antibody binding. To characterize the specificity of 2G12 more precisely, we performed solution-phase ELISA, carbohydrate microarray analysis, and cocrystallized Fab 2G12 with four different oligomannose derivatives (Man(4), Man(5), Man(7), and Man(8)) that compete with gp120 for binding to 2G12. Our combined studies reveal that 2G12 is capable of binding both the D1 and D3 arms of the Man(9)GlcNAc(2) moiety, which would provide more flexibility to make the required multivalent interactions between the antibody and the gp120 oligomannose cluster than thought previously. These results have important consequences for the design of immunogens to elicit 2G12-like neutralizing antibodies as a component of an HIV vaccine.

Fig. 1.

Chemical structure of Man₉GlcNAc₂ 1 and oligomannoses 2-9. The individual sugar residues of Man₉GlcNAc₂ are labeled in red. Elsewhere, all mannose residues of oligomannoses 2-9 are labeled to correspond with their structural equivalent on Man₉GlcNAc₂.

Scheme 1.

Scheme 2.

Fig. 2.

Analysis of 2G12 Ab binding to oligomannoses. (Left) ELISA of Oligomannose inhibition (%) of 2G12 binding to gp120 coated in microtiter plates. Black and gray bars represent the level of inhibition at oligomannose concentrations of 0.5 and 2.0 mM, respectively. (Right) Covalent oligomannose arrays in microtiter plates for analysis of 2G12 binding.

Fig. 3.

Crystal structure of Fab 2G12 with Man₄. (A)2F_o - F_c electron density map of the Man₄ sugar (shown in ball-and-stick) contoured at 1.8σ. Mannose residues are labeled in red. The light and heavy chains of Fab 2G12 are shown in cyan and purple. (B) Overlap of the Man₄ sugar (shown in blue) with a previous Fab 2G12 structure (7) bound to Man₉GlcNAc₂ (shown in green). The Man₄ clearly adopts a conformation similar to the D1 arm of Man₉GlcNAc₂. (C) Additional H bonds between Fab 2G12 and mannose 4 of Man₄. Two of the H bonds are water mediated (water molecules shown as gray spheres). These interactions may explain in the preference for D1 arm binding in the context of the full Man₉GlcNAc₂ moiety. Figures were made by using molscript (42), bobscript (43), and raster3d (44).

Fig. 4.

2G12 interactions with Man₅ and Man₈. (A)2F_o - F_c electron density map of the Man₅ sugar (shown in ball-and-stick) bound at the primary combining site of 2G12 contoured at 0.8σ. (B) Crystal structure of the domain-swapped Fab 2G12 dimer with Man₈. The light chains are shown in cyan, and the heavy chains are shown in red and purple. One Man₈ sugar (shown in ball-and-stick) is bound at each primary combining site of the domain-swapped Fab dimer, although in one site the sugar is bound by the D1 arm, whereas in the other the sugar is bound by the D3 arm. The D1 and D3 “arms” of the Man₈ sugar are labeled. (C) 2F_o - F_c electron density map of the Man₈ sugar (shown in ball-and-stick) contoured at 1.5σ. The Man₈ sugar cross-links the crystal together, with a D1 arm bound in the primary combining site of one Fab molecule and the D3 arm bound in the primary combining site of a different, crystallographically related Fab molecule. Figures were made by using molscript (42), bobscript (43), and raster3d (44).

Fig. 5.

Overlap of bound oligomannoses in six cocrystal structures with 2G12. The Man₄, Man₇, Man₈, and previously described Man₉GlcNAc₂ structures (shown as ball-and-stick in various shades of gray) all show 2G12 binding to the Manα1-2Manα1-2Man motif. The Man₅ and Man₈ structures (shown as ball- and-stick in shades of red) also show 2G12 binding to the Manα1-2Manα1-6Man motif, although only the two terminal sugars make contact. Full details of the Manα1-2Man interactions can be found in ref. . The figure was made by using molscript (42) and raster3d (44).