Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions.
The broadly neutralizing anti-HIV antibody 4E10 recognizes an epitope very close to the virus membrane on the glycoprotein gp41. It was previously shown that epitope recognition improves in a membrane context and that 4E10 binds directly, albeit weakly, to lipids. Furthermore, a crystal structure of Fab 4E10 complexed to an epitope peptide revealed that the centrally placed, protruding H3 loop of the antibody heavy chain does not form peptide contacts. To investigate the hypothesis that the H3 loop apex might interact with the viral membrane, two Trp residues in this region were substituted separately or in combination with either Ala or Asp by site-directed mutagenesis. The resultant IgG variants exhibited similar affinities for an epitope peptide as WT 4E10 but lower apparent affinities for both viral membrane mimetic liposomes and Env(-) virus. Variants also exhibited lower apparent affinities for Env(+) virions and failed to significantly neutralize a number of 4E10-sensitive viruses. For the extremely sensitive HXB2 virus, variants did neutralize, but at 37- to >250-fold lower titers than WT 4E10, with Asp substitutions exerting a greater effect on neutralization potency than Ala substitutions. Because reductions in lipid binding reflect trends in neutralization potency, we conclude that Trp residues in the antibody H3 loop enable membrane proximal epitope recognition through favorable lipid interactions. The requirement for lipophilic residues such as Trp adjacent to the antigen binding site may explain difficulties in eliciting 4E10-like neutralizing antibody responses by immunization and helps define a unique motif for antibody recognition of membrane proximal antigens.
Scherer
Erin M
EM
Department of Immunology, University of Washington, Seattle, WA 98195, USA.
Leaman
Daniel P
DP
Zwick
Michael B
MB
McMichael
Andrew J
AJ
Burton
Dennis R
DR
D000595:Amino Acid Sequence; D057134:Antibodies, Neutralizing / Q000276:immunology; D001665:Binding Sites; D002462:Cell Membrane / Q000276:immunology*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006678:HIV / Q000276:immunology*; D015483:HIV Antibodies / Q000276:immunology*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D008563:Membrane Lipids / Q000737:chemistry / Q000276:immunology; D008969:Molecular Sequence Data
D057134:Antibodies, Neutralizing; D000939:Epitopes; D015483:HIV Antibodies; D015700:HIV Envelope Protein gp41; D007074:Immunoglobulin G; D008563:Membrane Lipids