NMR MAPPING OF THE ANTIGENIC DETERMINANT RECOGNIZED BY AN ANTI-GP120,HUMAN-IMMUNODEFICIENCY-VIRUS NEUTRALIZING ANTIBODY

The 24-amino-acid peptide RP135 (NNTRKSIRIQRGPGRAFVTIGKIG) corresponds in its amino acid sequence to the principal neutralizing determinant of the human immunodeficiency virus type-1, IIIB isolate (HIV-1(IIIb), residues 308-331 of the envelope glycoprotein gp120). In order to map the antigenic determinant recognized by 0.5 beta, the complex of RP13 5 with an anti-gp120 HIV neutralizing antibody, 0.5 beta, which cross reacts with the peptide, was studied by using two-dimensional NMR spec troscopy. A combination of homonuclear Hartmann Hahn two-dimensional e xperiment and rotating-frame Overhauser enhancement spectroscopy of th e Fab/peptide complex measured in H2O was used to eliminate the resona nces of the Fab and the tightly bound peptide residues and to obtain s equential assignments for those parts of the peptide which retain cons iderable mobility upon binding. In this manner, a total of 14 residues (Ser6-Thr19) were shown to be part of the antigenic determinant recog nized by the antibody 0.5 beta. Lys5 and Ile20 were found to retain co nsiderable mobility in the bound peptide while their amide protons und ergo significant change in chemical shift upon binding. This observati on suggests that these two residues are at the boundaries of the deter minant recognized by the antibody. Competitive binding experiments usi ng truncated peptides strongly support the NMR observations.