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Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions.

ABSTRACT: 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.

SUBMITTER: Scherer EM

PROVIDER: S-EPMC2824387 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions.

Scherer Erin M EM Leaman Daniel P DP Zwick Michael B MB McMichael Andrew J AJ Burton Dennis R DR

Proceedings of the National Academy of Sciences of the United States of America 20100104 4

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 l ...[more]

PMID: 20080706

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Project description:Mature antibodies (Abs) that are exquisitely specific for virtually any foreign molecule may be produced by affinity maturation of naïve (or germline) Abs. However, the finite number of germline Abs available suggests that, in contrast to mature Abs, germline Abs must be broadly polyspecific so that they are able to recognize a wide range of ligands. Thus, affinity maturation must play a role in mediating Ab specificity. One biophysical property that distinguishes polyspecificity from specificity is protein flexibility; a flexible combining site is able to adopt different conformations that recognize different foreign molecules (or antigens), while a rigid combining site is locked into a conformation that is specific for a given antigen. Recent studies (Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8821-8826) have examined, at the atomic level, the structural properties that mediate changes in flexibility at four stages of affinity maturation in the 4-4-20 Ab. These studies employed molecular dynamics simulations to reveal a network of residue interactions that mediate the flexibility changes accompanying maturation. The flexibility of the Ab combining sites in these molecular systems was originally measured using three-pulse photon echo spectroscopy (3PEPS). The present investigation extends this work by providing a concrete link between structural properties of the Ab molecules and features of the spectroscopic measurements used to characterize their flexibility. Results obtained from the simulations are in good qualitative agreement with the experimental measurements and indicate that the spectroscopic signal is sensitive to protein dynamics distributed throughout the entire combining site. Thus, the simulations provide a molecular-level interpretation of the changes induced by affinity maturation of the Ab. The results suggest that 3PEPS spectroscopy in combination with molecular dynamics simulations can provide a detailed description of protein dynamics and, in this case, how it is evolved for biological function.

Dataset Information

Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions.

Publications

Aromatic residues at the edge of the antibody combining site facilitate viral glycoprotein recognition through membrane interactions.

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