Antibody Domain Exchange Is an Immunological Solution to Carbohydrate Cluster Recognition

Abstract

Human antibody 2G12 neutralizes a broad range of human immunodeficiency virus type 1 (HIV-1) isolates by binding an unusually dense cluster of carbohydrate moieties on the "silent" face of the gp120 envelope glycoprotein. Crystal structures of Fab 2G12 and its complexes with the disaccharide Manalpha1-2Man and with the oligosaccharide Man9GlcNAc2 revealed that two Fabs assemble into an interlocked VH domain-swapped dimer. Further biochemical, biophysical, and mutagenesis data strongly support a Fab-dimerized antibody as the prevalent form that recognizes gp120. The extraordinary configuration of this antibody provides an extended surface, with newly described binding sites, for multivalent interaction with a conserved cluster of oligomannose type sugars on the surface of gp120. The unique interdigitation of Fab domains within an antibody uncovers a previously unappreciated mechanism for high-affinity recognition of carbohydrate or other repeating epitopes on cell or microbial surfaces.

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

“…Human MAb 2G12 Fab was produced as before (11). The 2G12.1 sequence was synthesized as a peptide (sequence: NH 3 -ACPPSHVLDMRSGTCLAAEGK(biotin)-NH 2 ) by Multiple Peptide Synthesis (San Diego, CA, USA).…”

“…The close packing of Fv domains leads to multivalent carbohydrate binding at up to four sites: two within each Fv domain and two in the interface between the exchanged V H regions (11,41). Because of V H exchange, 2G12 Fabs are dimeric (i.e., comprising 2 V H -C H 1 chains and 2 light chains).…”

“…2G12 also reacts with monosaccharides, such as mannose and fructose, with much lower affinity (8,11). To assess whether 2G12.1 peptide interacts with the carbohydrate-binding sites of MAb 2G12, competition assays were performed between 2G12.1 peptide and a variety of carbohydrates.…”

“…The crystal structure of MAb 2G12 in complex with a synthetic peptide (2G12.1) was compared with previously published structures of 2G12 in complex with Man 9 GlcNAc 2 and Manα1-2Man (11,15). The 2G12-bound peptide exhibited minimal spatial overlap with the bound oligosaccharides, and common contacts with the antibody were limited to a few residues, which reveals that the mechanism of antibodypeptide recognition differs from that for the oligomannose epitope on gp120.…”

“…MAb 2G12 binds with high affinity to a unique, conserved epitope on the HIV-1 envelope that is formed by a cluster of N-linked, high-mannose glycan groups on the "silent" face of gp120 (7)(8)(9)(10). Crystal structures of 2G12 Fab alone, and in complex with oligosaccharides Man 9 GlcNAc 2 and Manα1-2Man, revealed that two Fab monomers assemble into an interlocked, domain-swapped dimer, that forms an extensive, multivalent binding surface (11). Four Man 9 GlcNAc 2 moieties were observed bound to the Fab dimer in the crystal structure, occupying the two canonical antigen-binding sites, and two novel ones located at the assembled interface of the two interlocked V H domains.…”