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| Abstract |
The 4E10 antibody displays an extreme breadth of HIV-1 neutralization and therefore constitutes a suitable model system for structure-guided vaccine design and immunotherapeutics against AIDS. In this... regard, the relevance of autoreactivity with membrane lipids for the biological function of this antibody is still a subject of controversy. To address this dispute, herein we have compared the membrane partitioning ability of the 4E10 antibody and several of its variants, which were mutated at the region of the paratope surface in contact with the membrane interface. We first employed a physical separation approach (vesicle flotation) and subsequently carried out quantitative fluorescence measurements in an intact system (spectroscopic titration), using 4E10 Fab labeled with a polarity-sensitive fluorescent probe. Moreover, recognition of epitope peptide in membrane was demonstrated by photo-cross-linking assays using a Fab that incorporated the genetically encoded unnatural amino acid p-benzoylphenylalanine. The experimental data ruled out that the proposed stereospecific recognition of viral lipids was necessary for the function of the antibody. In contrast, our data suggest that nonspecific electrostatic interactions between basic residues of 4E10 and acidic phospholipids in the membranes contribute to the observed biological function. Moreover, the energetics of membrane partitioning indicated that 4E10 behaves as a peripheral membrane protein, tightening the binding to the ligand epitope inserted in the viral membrane. The implications of these findings for the natural production and biological function of this antibody are discussed.show more
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