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Abstract
The construction, expression, and purification of an active Fv fragment of the 0.5beta monoclonal human immunodeficiency virus type 1 (HIV-1) neutralizing antibody is reported. The interaction between the Fv fragment and the RP135 peptide derived from the V3 loop of gp120 from HIV-1IIIB was studied by varying the salt concentration and by mutating arginine residues in the peptide. The mutations R4A, R8A and R11A (which correspond to residues 311, 315, and 318 in gp120 of HIV-1IIIB) reduce the binding free energy by 0.22 (+/- 0. 20), 4.32 (+/- 0.16), and 1.58 (+/- 0.17) kcal mol-1, respectively. The salt-dependent components of their contributions to binding are 0.02 (+/- 0.22), -0.55 (+/- 0.18), and -0.97 (+/- 0.19) kcal mol-1, respectively. The magnitudes of the mutational effects and the extent of shielding by 1 M NaCl suggest that Arg-8 is involved in a buried salt bridge in the peptide-Fv fragment complex, whereas Arg-11 is involved in a more solvent-exposed electrostatic interaction.
Highlights
Nuclear magnetic resonance (NMR) studies on the interaction of the RP135 peptide with the Fab fragment of the 0.5 antibody have defined a 16-residue epitope from Lys-5 to Ile-20 [5]
The recently solved crystal structure of the Fab fragment of a different human immunodeficiency virus type 1 (HIV-1) neutralizing antibody, 50.1, in complex with a 16-residue peptide derived from the V3 loop of gp120 (HIV-1MN strain) shows that it interacts only with a sequential 7-residue epitope [6]
We have focused on arginine residues in the peptide since amino acid sequence information and model building [5] suggested that electrostatic interactions are of special importance in this system
Summary
Nuclear magnetic resonance (NMR) studies on the interaction of the RP135 peptide with the Fab fragment of the 0.5 antibody have defined a 16-residue epitope from Lys-5 to Ile-20 [5]. A 24-amino acid-long peptide, NNTRKSIRIQRGPGRAFVTIGKIG, derived from the principal neutralizing determinant of gp120 of HIV-1IIIB and designated RP135, is immunogenic by itself and was shown to correspond to the binding site of gp120 to the 0.5 antibody [4].
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