A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120

Abstract

MAb 2G12 neutralizes HIV-1 by binding with high affinity to a cluster of high-mannose oligosaccharides on the envelope glycoprotein, gp120. Screening of phage-displayed peptide libraries with 2G12 identified peptides that bind specifically, with K(d)s ranging from 0.4 to 200 microM. The crystal structure of a 21-mer peptide ligand in complex with 2G12 Fab was determined at 2.8 A resolution. Comparison of this structure with previous structures of 2G12-carbohydrate complexes revealed striking differences in the mechanism of 2G12 binding to peptide vs. carbohydrate. The peptide occupies a site different from, but adjacent to, the primary carbohydrate-binding site on 2G12, and makes only slightly fewer contacts to the Fab than Man(9)GlcNAc(2) (51 vs. 56, respectively). However, only two antibody contacts with the peptide are hydrogen bonds in contrast to six with Man(9)GlcNAc(2), and only three of the antibody residues that interact with Man(9)GlcNAc(2) also contact the peptide. Thus, this mechanism of peptide binding to 2G12 does not support structural mimicry of the native carbohydrate epitope on gp120, since it neither replicates the oligosaccharide footprint on the antibody nor most of the contact residues. Moreover, 2G12.1 peptide is not an immunogenic mimic of the 2G12 epitope, since antisera produced against it did not bind gp120.

Figure 1

Binding of 2G12 MAb to 2G12.1 peptide. A) Titration ELISA of 2G12 IgG and Fab on 2G12.1 peptide captured with streptavidin. Bound antibodies were detected with a goat anti-human antibody conjugated to alkaline phosphatase and pNPP. Values are expressed as optical density at 405 nm. B) Titration of 2G12.1 peptide by capture from solution with plate-adsorbed MAb 2G12. Bound peptide was detected with neutravidin-horseradish peroxidase and ABTS. Values are expressed as optical density at 405 − 490 nm. One representative experiment of two is shown.

Figure 2

Inhibition of 2G12 binding to 2G12.1 peptide (A) and gp120 (B) in the presence of monosaccharides and 2G12.1 peptide. Binding of 2G12 was assayed after preincubation of 1 or 100 nM IgG (for gp120 and peptide binding assay, respectively) with serial dilutions of glucose, mannose, fructose, and 2G12.1 peptide. Antibody binding in the absence of a competitor was considered 100%; values are expressed as percent binding.

Figure 3

Alanine-substitution scanning of the 2G12.1 peptide in the context of the phage. The relative binding of 20 nM (A) and 5 nM (B) 2G12 IgG to the alanine-substituted phage is expressed as percentage binding of each mutant phage with respect to wild-type (wt) 2G12.1 phage. The f88 is a negative control for wild-type phage expressing no recombinant peptide. One representative experiment of two is shown.

Figure 4

A) Overall crystal structure of the Fab 2G12 dimer bound to the biotinylated 21-mer 2G12.1 peptide. Each Fab of the domain-swapped dimer interacts with 2G12.1 peptide (shown in a yellow ball-and-stick representation), only on one side of the primary carbohydrate-binding site. The light chains are shown in cyan; the heavy chains from the individual Fabs in the dimer are shown in red and purple for clarity. The CDR L1, L2, L3, H1, H2, and H3 loops are labeled and colored orange, pink, green, blue, purple, and yellow, respectively. This color scheme is followed throughout all the subsequent figures. B) Top view of the 2G12.1 peptide-Fab complex, looking down onto the combining site, asterisk indicates the binding site for oligosaccharide antigens. C) Side view of the Fab-bound 2G12.1 peptide.

Figure 5

Stereoview of 2G12.1 peptide electron density. The final 2Fo-Fc electron density for the peptide after refinement, with the final peptide coordinates, is contoured at 1.8σ.

Figure 6

Hydrogen bonding in the 2G12.1-2G12 structure. A) Stereoview of the 2G12.1 peptide with intrapeptide hydrogen bonds represented as black dotted lines. The peptide forms a 2-stranded β-sheet. B) Schematic representation of direct contacts between 2G12.1 peptide and Fab. Black lines represent 1–4 van der Waals' interactions between residues, green lines represent 5–10 van der Waals' interactions, and red lines represent hydrogen bonds. Peptide residues shown to be critical for binding to 2G12 by alanine mutagenesis are indicated by an asterisk, and other important residues are indicated by a plus sign. Fab residue Gly ^L93 (L3), which contacts both Man₉GlcNAc₂ and 2G12.1 peptide, is underlined. Tyr^H56 (H2) may also contact Man₉GlcNAc₂ (see text for details). Note that the hydrogen bond Ala^P1-2G12 D^H58 is only observed in two of the four MAb-peptide complexes, and therefore is not shown here.

Figure 7

A) Hydrogen bond between 2G12.1 Ala^P1 with 2G12 D^H58 (H2). B) Modeled hydrogen bonds between 2G12.1-KH3 peptide Ser^P1 with 2G12 D^H58 (H2) and Tyr^H56 (H2). Dotted black lines represent hydrogen bonds. C) Binding of 2G12 IgG to 2G12.1 phage clone and to optimized derivatives. Values are expressed as optical density at 405 − 490 nm.

Figure 8

A) Overlap of the Man₉GlcNAc₂-Fab 2G12 and peptide 2G12.1-Fab 2G12 structures. The Man₉GlcNAc₂ glycan is shown in red, while the 2G12.1 peptide is shown in blue. The 2G12.1 peptide sterically clashes with the D2 and D3 arms of the Man₉GlcNAc₂ moiety, illustrating why this peptide is capable of competing with this glycan for 2G12 binding. B) Molecular surface representation of a 2G12 monomer showing antibody areas in contact with Man₉GlcNAc₂, (cyan shading) and with 2G12.1 peptide (black outline). The Man₉GlcNAc₂ is shown in a yellow ball-and-stick representation, and the 2G12.1 peptide is shown as a red Cα tube. C) Same as in B, but the surface in contact with Manα1-2Man is shown in cyan, with its sugar coordinates. PCBS refers to the primary carbohydrate-binding site, as defined in Calarese et al. (11, 15).