Structure of a high-affinity "mimotope" peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies

Abstract

The human antibody b12 recognizes a discontinuous epitope on gp120 and is one of the rare monoclonal antibodies that neutralize a broad range of primary human immunodeficiency virus type 1 (HIV-1) isolates. We previously reported the isolation of B2.1, a dimeric peptide that binds with high specificity to b12 and competes with gp120 for b12 antibody binding. Here, we show that the affinity of B2.1 was improved 60-fold over its synthetic-peptide counterpart by fusing it to the N terminus of a soluble protein. This affinity, which is within an order of magnitude of that of gp120, probably more closely reflects the affinity of the phage-borne peptide. The crystal structure of a complex between Fab of b12 and B2.1 was determined at 1.8 A resolution. The structural data allowed the differentiation of residues that form critical contacts with b12 from those required for maintenance of the antigenic structure of the peptide, and revealed that three contiguous residues mediate B2.1's critical contacts with b12. This single region of critical contact between the B2.1 peptide and the b12 paratope is unlikely to mimic the discontinuous key binding residues involved in the full b12 epitope for gp120, as previously identified by alanine scanning substitutions on the gp120 surface. These structural observations are supported by experiments that demonstrate that B2.1 is an ineffective immunogenic mimic of the b12 epitope on gp120. Indeed, an extensive series of immunizations with B2.1 in various forms failed to produce gp120 cross-reactive sera. The functional and structural data presented here, however, suggest that the mechanism by which b12 recognizes the two antigens is very different. Here, we present the first crystal structure of peptide bound to an antibody that was originally raised against a discontinuous protein epitope. Our results highlight the challenge of producing immunogens that mimic discontinuous protein epitopes, and the necessity of combining complementary experimental approaches in analyzing the antigenic and immunogenic properties of putative molecular mimics.

Figure 1

Alanine-substitution scanning of the B2.1 sequence. (a) Binding of IgG b12 (grey bars) and Fab b12 (black bars) to phage displaying Ala-substituted B2.1 sequences. (b) Binding of sera from each of three mice (grey, black, and white bars) immunized with B2.1 synthetic peptide-OVA conjugate. Results are expressed as % binding of each mutant phage with respect to wild-type (wt) B2.1 phage; f88 is a negative control for phage bearing only wild-type pVIII protein. *Ala replacement of the Cys residue was not included in this study, as previous work showed substitution of this Cys by Ser abrogates b12 binding.

Figure 2

Crystal structure of the b12-B2.1 complex. (a) the B2.1 peptide comprises a homodimer formed by chains P (green) and R (yellow). Chain P (green) is bound to Fab LH, which is designated in the deposited PBD coordinates by heavy chain H (blue) and light chain L (red). Chain R (yellow) is bound to Fab MK, which is designated by heavy chain K (light blue) and light chain M (light pink). (b) Each of the two chains of the B2.1 peptide independently contacts one of the two b12 Fabs in the asymmetric unit. Key side chains of B2.1 are illustrated in ball-and-stick. The extended CDR H3s of the b12 antibody reach across each peptide monomer and do not contact the opposing chain. (c) Bound and unbound structures of Fab b12 are superimposed. The two unliganded Fabs from the IgG structure are colored grey, whereas the two B2.1-bound Fabs are colored as in panels a and b. Only those side chains that adopt conformers different between free and bound Fab structures are represented. Trp H100 adopts similar conformers, but varies up to 8Å in position because of the 1-3 Å adjustment of the CDRH3 main chain.

Figure 3

B2.1-b12 interactions. In all panels, only one chain (P) of the B2.1 peptide is shown bound to its respective Fab b12 (LH), with the light chain colored pink and the heavy chain blue in panels a-d. (a) Top view of the Fab b12 antigen-binding site with the bound B2.1 peptide chain illustrated in yellow ball-and-stick representation with the N and C termini of the chain indicated. The b12 combining site is illustrated as a molecular surface with the locations of the six CDRs (L1, L2, L3, H1, H2, and H3) indicated. (b)Side view of the b12 antibody-combining site. The B2.1 chain (P) dips into a canyon formed between CDRs L1, L3 and H3. (c) Specific contacts between B2.1 chain (P) and the Fab light chain (L) involve the residues Ser 8, Asp 9 and Leu 10 of the originally-selected, SDLX₃CI consensus motif. Main chain carbonyls are not pictured for clarity. (d) Side view of the b12 antibody-combining site illustrating interactions of the B2.1 chain (P) (yellow) with b12 heavy chain (H) residues Pro H100d, Gln H100e, Trp 50, Asn H56, Lys 57, and Glu H58. Heavy chain contact residues are illustrated in blue in ball and stick representation. (e) Molecular surface representation of b12 showing the antibody areas important for binding to B2.1 and b12. Shaded areas indicate substitutions that: (i) decrease binding to both B2.1 and gp120, red; (ii) have opposite effects on B2.1 and gp120 binding, red and white striped; (iii) affect gp120 binding only, blue; (iv) affect B2.1 binding only, green; and (v) have no effect on either antigen, black. Mutagenesis data are from Zwick et al. (11).

Figure 4

Schematic representation of direct contacts between one B2.1 peptide chain, R and b12 Fab, MK (top panel), and contacts between the two chains of a single B2.1 dimer (bottom panel). Atomic contact data are taken from Supplementary Table 2. Red lines represent hydrophobic contacts, blue lines represent hydrogen bonds, blue dashed lines represent interactions through a water molecule, and the dotted black line represents the disulfide bridge within each B2.1 molecule. Thinner lines represent a single pair of contacting atoms whereas thicker lines represent 2-4 atomic contacts per residue. Peptide residues critical for binding to b12 are indicated by *; residues important (but not critical) for binding are indicated by +, and nd is not determined.