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

Abstract

As part of a program to study the effect of glycosylation on the three-dimensional structures of HIV-ImB V3 peptide constructs, we have examined the solution structures of a 15 residue peptide (RIQRGPGRAFVTIGK, P18ms), originally mapped as an epitope recognized by CD8 + D u class I MHC-restricted murine cytotoxic T-lymphocytes (CTL), and an analogue (P18mB-g), O-glycosylated with an t~-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 (GS-RS). Glycosylation on T Iz '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 Tlymphocytes (CTL) to lyse tumor cells presenting P18ttm was completely abrogated by threonine glycosylation.

Conformational influences of glycosylation of a peptide: A possible model for the effect of glycosylation on the rate of protein folding

“…In principle other noninteractive, noncarbohydrate moieties mounted at the ␥ carboxyl of the asparganine residue might well have induced their own conformational restrictions on the peptide. However, our work in concert with earlier findings (14)(15)(16)(17)(18)(19)(20)(21) tends to suggest the existence of such specific contacts as biasing element in glycopeptides. It has been argued theoretically that a modest change in biasing the energy landscape can have a significant effect on reducing the number of folding paths a protein may choose with concomitant increase in the rate of productive folding (3).…”

“…In the case of O-linked glycopeptides (14,15,20), NMR studies have shown that the peptide backbone responds to glycosylation, as evidenced by changes in sequential amideamide nuclear Overhauser effect (NOE) interactions. The response is further modulated by whether the sugar component is a mono-or disaccharide.…”

“…However, in trying to determine the extent to which glycosylation influences early stages of the protein folding process, glycopeptides provide more appropriate models since the conformation of the system is not dominated by the overall fold of the ultimately organized protein structure. Some recent studies on glycopeptides have provided insights into potential conformational consequences of glycosylation for both .In the case of O-linked glycopeptides (14,15,20), NMR studies have shown that the peptide backbone responds to glycosylation, as evidenced by changes in sequential amideamide nuclear Overhauser effect (NOE) interactions. The response is further modulated by whether the sugar component is a mono-or disaccharide.…”

Crystal Structures of Human Immunodeficiency Virus Type 1 (HIV-1) Neutralizing Antibody 2219 in Complex with Three Different V3 Peptides Reveal a New Binding Mode for HIV-1 Cross-Reactivity

“…NMR studies of isolated V3 peptides show no stable structure in solution, but transient turns exist around GPGR (7-9, 17, 18, 19, 25, 33, 36, 37, 52, 64, 77, 79, 80, 84). NMR studies of peptides modified by cyclization (4,9,33,38,74,(76)(77)(78), by replacement of Ala P316 with the conformationally restricted residue ␣-aminoisobutyric acid (4,25), by glycosylation (36,37,52), through attachment to resin beads (40), through attachment to a bacteriophage viral coat protein (39), and through attachment to carrier proteins, such as bovine pancreatic trypsin inhibitor (81) and MUC1 (22), all show an increased ␤-turn propensity around GP GRAF, while V3 peptides attached to filamentous bacteriophage fd viral coat protein pVIII (39) adopt a double-turn structure similar to that observed in the Fab 59.1-peptide crystal structure (25,26).…”

The Binding of a Glycoprotein 120 V3 Loop Peptide to HIV-1 Neutralizing Antibodies

“…These methods included aminoisobutyric acid substitution (10), insertion into a viral coat protein (23), glycosylation (12)(13)(14), attachment to resin beads (24), cyclization of the peptide (15), and trifluoroethanol addition (14 -19).…”