Structure-affinity relationships in the gp41 ELDKWA epitope for the HIV-1 neutralizing monoclonal antibody 2F5: effects of side-chain and backbone modifications and conformational constraints. |
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Authors: | Y Tian C V Ramesh X Ma S Naqvi T Patel T Cenizal M Tiscione K Diaz T Crea E Arnold G F Arnold J W Taylor |
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Institution: | Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, USA; also Center for Advanced Biotechnology and Medicine, Piscataway, NJ 08854, USA. |
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Abstract: | The human monoclonal antibody, mAb 2F5, has broad HIV-1 neutralizing activity and binds a conserved linear epitope within the envelope glycoprotein gp41 having a core recognition sequence ELDKWA. In this study, the structural requirements of this epitope for high-affinity binding to mAb 2F5 were explored using peptide synthesis and competitive enzyme-linked immunosorbant assay (ELISA). Expansion of the minimal epitope to an end-capped, linear nonapeptide, Ac-LELDKWASL-amide, was sufficient to attain maximal affinity within the set of native gp41-sequence peptides assayed. Scanning single-residue alanine and d-residue substitutions then confirmed the essential recognition requirements of 2F5 for the central DKW sequence, and also established the importance of the terminal leucine residues in determining high-affinity binding of the linear nonapeptide. Further studies of side-chain and backbone-modified analogs revealed a high degree of structural specificity for the DK sequence in particular, and delineated the steric requirements of the Leu(3) and Trp(6) residues. The nine-residue 2F5 epitope, flanked by pairs of serine residues, retained a high affinity for 2F5 when it was conformationally constrained as a 15-residue, disulfide-bridged loop. However, analogs with smaller or larger loop sizes resulted in lower 2F5 affinities. The conformational effects of the gp41 C-peptide helix immediately adjacent to the N-terminal end of the ELDKWA epitope were examined through the synthesis of helix-initiated analogs. Circular dichroism (CD) studies indicated that the alpha-helical conformation was propagated efficiently into the LELDKWASL epitope, but without any significant effect on its affinity for 2F5. This study should guide the design of a second generation of conformationally constrained ELDKWA analogs that might elicit an immune response that mimics the HIV-neutralizing actions of 2F5. |
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