Structural comparison of a 15 residue peptide from the V3 loop of HIV‐1IIIb and an O‐glycosylated analogue

Abstract

As part of a program to study the effect of glycosylation on the three‐dimensional structures of HIV‐1 _IIIB V3 peptide constructs, we have examined the solution structures of a 15 residue peptide (RIQRGPGRAFVTIGK, P18 _IIIB ), originally mapped as an epitope recognized by CD8⁺ D ^d class I MHC‐restricted murine cytotoxic T‐lymphocytes (CTL), and an analogue (P18 _IIIB ‐g), O‐glycosylated with an α‐galactosamine on Thr‐12, using NMR, circular dichroism and molecular modeling methods. Our studies show that the peptides sample mainly random conformations in aqueous solution near 25°C and become more ordered by the addition of trifluoroethanol. Upon decreasing the temperature to 5°C, a reverse turn is formed around the immunodominant tip (G⁵−R⁸). Glycosylation on T¹² ‘tightens’ the turn slightly as suggested by NOE and CD analysis. In addition, the sugar has a defined conformation with respect to the peptide backbone and influences the local peptide conformation. These data suggest that simple glycosylation may influence the conformational equilibrium of a V3 peptide which contains a domain critical for antibody recognition and virus neutralization. We also show that the ability of cytotoxic T‐lymphocytes (CTL) to lyse tumor cells presenting P18 _IIIB was completely abrogated by threonine glycosylation.