Two Structural States of Complexes of Peptide and Class II Major Histocompatibility Complex Revealed by Photoaffinity-labeled Peptides

Eugenio Carrasco-Marı́N,Shirley Petzold,Emil R Unanue

doi:10.1074/jbc.274.44.31333

Eugenio Carrasco-Marı́N, Shirley Petzold + Show 1 more

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https://doi.org/10.1074/jbc.274.44.31333

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Abstract

The complex of the murine class II histocompatibility molecules I-A(k) with high affinity binding peptides were resistant to denaturation when examined by SDS-polyacrylamide gel electrophoresis at various pH levels. In contrast, complexes made with low affinity binding peptides were highly sensitive to denaturation by SDS. This effect was more pronounced at low pH. Placing a photoactivatable probe at the amino terminus of the peptides resulted in their covalent linkage to soluble I-A(k) molecules. We found an inverse relationship between the capacity of peptides to form SDS-stable complexes with I-A(k) and their extent of covalent association with either the alpha or beta chain. The relationship held true for three different peptides in which the main anchor residues were changed so as to affect their binding affinity for I-A(k) molecules. Thus, high affinity peptides generate a complex in which the motion of their amino termini was restricted, whereas complexes of low affinity peptides are more flexible. In agreement with this observation, complexes of I-A(k) with high affinity peptides were highly resistant to proteolysis, in contrast to those formed with weakly binding peptides, which were more likely to be cleaved. Complexes with low affinity peptides generate a structure with enhanced flexibility as compared with complexes with high affinity peptides.

Highlights

We have been examining the interaction of peptides with class II major histocompatibility complex (MHC)1 molecules, attempting to relate binding interactions to biological responses
Studying the I-Ak class II molecules, we found that peptides resulted in either stable or unstable complexes with I-Ak, i.e. SDSresistant or SDS-sensitive complexes [4, 5]. (The complexes of peptide-class II molecules resist or are sensitive to dissociation if subjected to the SDS-PAGE at room temperature, i.e. stable and unstable complexes, respectively; at 100 °C the components of the complex always dissociate.) The conditions that determined stable or unstable SDS complexes were related to the presence of high affinity binding peptides in which, first, a critical P1 anchor residue was represented by an appropriate amino acid side chain and, second, the polypeptide chain was of appropriate length (ϳ16 residues)
Most of the results shown in this study were made using the 48 – 61 peptide of hen egg white lysozyme (DGSTDYGILQINSR) bound to I-Ak, using variants in which the main anchor residue for the P1 pocket was changed in order to weaken the affinity of the peptide for the I-Ak class II molecule (i.e. Asp at 52)

Summary

Introduction

We have been examining the interaction of peptides with class II major histocompatibility complex (MHC) molecules, attempting to relate binding interactions to biological responses. (The complexes of peptide-class II molecules resist or are sensitive to dissociation if subjected to the SDS-PAGE at room temperature, i.e. stable and unstable complexes, respectively; at 100 °C the components of the complex always dissociate.) The conditions that determined stable or unstable SDS complexes were related to the presence of high affinity binding peptides in which, first, a critical P1 anchor residue was represented by an appropriate amino acid side chain (with I-Ak, that of aspartic acid or glutamine) and, second, the polypeptide chain was of appropriate length (ϳ16 residues). We examined peptides in which we changed this P1 anchor residue from aspartic acid to other amino acids that reduced their binding strength and SDS-PAGE behavior These peptides were bound to I-Ak, and the complexes were tested, first, for the effects of the pH on their SDS resistance or susceptibility and, second, for their interaction using photoactivatable 48 – 61 peptides. The I-Ak peptide complexes resistant to SDS denaturation are likewise highly resistant to proteolytic enzymes, in contrast to those sensitive to SDS denaturation

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