Characterization of class I MHC folding intermediates and their disparate interactions with peptide and β2-microglobulin

Abstract

Newly synthesized class I heavy chains achieve domain structure using disulfide bonds, assemble with beta-2 microglobulin ( β 2m), and bind peptide ligand to complete the trimeric complex. Although each of these initial events is thought to be critical for class I folding, their sequential order and effect on class I structure are unknown. Using mAb specific for distinct conformations of H-2L d and L q, we have defined folding intermediates of class I molecules. We show here that non-peptide-associated forms of L d or L q, detected by mAb 64-3-7 and designated L alt, lack numerous conformational epitopes surrounding their ligand binding sites. These results support the notion that L alt molecules have an open conformation. Interestingly, a significant proportion of L alt molecules were detected in association with β 2m and these L alt β 2m heterodimers were preferentially folded by peptide in cell lysates. These findings indicate that class I heavy chain β 2m association can precede ligand binding and that peptide is probably the limiting factor for completion of the L d/β 2m/peptide trimeric complex in vivo. The characteristics of L alt molecules were investigated further by ascertaining the disulfide bond status of these molecules and their association with β 2m and peptide. Treatment of cells with dithiothreitol (DTT), a membrane-permeable reducing agent, demonstrated that L alt molecules constitute a heterogeneous population including reduced, partially reduced, partially reduced and native class I molecules. Furthermore, partially reduced L d alt molecules, in a cell line expressing a mutant L d molecule lacking the α2 domain disulfide bond, accumulated intracellularly, were not β 2m-associated and displayed marginal peptide-induced folding in vitro. In accordance with this latter finding, peptide was found to preferentially convert fully disulfide-bonded forms of L d alt to conformed L d. Thus, we propose that intrachain disulfide bond formation precedes the association of class I heavy chain with β 2m and peptide, and that disulfide bond formation is required for efficient assembly, ligand binding and folding of the class I heavy chain.