The major histocompatibility complex (MHC) class I molecule plays a crucial role in cytotoxic lymphocyte function. Functional class I MHC exists as a heterotrimer consisting of the MHC class I heavy chain, an antigenic peptide fragment, and beta2-microglobulin (beta2m). beta2m has been previously shown to play an important role in the folding of the MHC heavy chain without continued beta2m association with the MHC complex. Therefore, beta2m is both a structural component of the MHC complex and a chaperone-like molecule for MHC folding. In this study we provide data supporting a model in which the chaperone-like role of beta2m is dependent on initial binding to only one of the two beta2m interfaces with class 1 heavy chain. beta2-Microglobulin binding to an isolated alpha3 domain of the class I MHC heavy chain accurately models the biochemistry and thermodynamics of beta2m-driven refolding. Our results explain a 1000-fold discrepancy between beta2m binding and refolding of MHC1. The biochemical study of the individual domains of complex molecules is an important strategy for understanding their dynamic structure and multiple functions.
The major histocompatibility complex class I (MHC1) molecule plays a crucial role in cytotoxic lymphocyte function. 2-Microglobulin (2m) has been demonstrated to be both a structural component of the MHC1 complex and a chaperone-like molecule for MHC1 folding. 2m binding to an isolated ␣3 domain of MHC1 heavy chain at micromolar concentrations has been shown to accurately model the biochemistry and thermodynamics of 2m-driven MHC1 folding. These results suggested a model in which the chaperone-like role of 2m is dependent on initial binding to the ␣3 domain interface of MHC1 with 2m. Such a model predicts that a mutant 2m molecule with an intact MHC1 ␣3 domain interaction but a defective MHC1 ␣1␣2 domain interaction would block 2m-driven folding of MHC1. In this study we generated such a 2m mutant and demonstrated that it blocks MHC1 folding by normal 2m at the expected micromolar concentrations. Our data support an initial interaction of 2m with the MHC1 ␣3 domain in MHC1 folding. In addition, the dominant negative mutant 2m can block T-cell functional responses to antigenic peptide and MHC1.The major histocompatibility complex class I (MHC1) 1 molecule and antigenic peptide are recognized by CD8ϩ cytotoxic T-lymphocytes (CTL) in CTL activation and lysis of targets (1). The heavy chain of the MHC1 molecule can interact noncovalently with a number of other molecules in the formation of a CTL activating complex. These include the MHC1 light chain or 2m, the antigenic peptide fragment, the T-cell receptor (TCR), and the CD8 molecule (2). The specificity of the CTL response resides in the selective MHC1 binding of specific antigenic peptide fragments and in the TCR recognition of these antigenic peptides and MHC1 (3, 4). The MHC1 contact surface for TCR and peptide binding is formed by the ␣1 and ␣2 domains of the three-domain MHC1 heavy chain (2, 5-7).The MHC1 heavy chain ␣1 and ␣2 domains, as well as the immunoglobulin-like ␣3 domain, have been shown by x-ray crystallography (8) to interact with 2m, the nonpolymorphic component of the MHC1 complex. Mutations in the ␣1 (9, 10) or ␣3 (11) domains of the MHC1 heavy chain lead to changes in 2m binding. These studies demonstrate that the functional interaction of the MHC1 heavy chain with 2m occurs at multiple surfaces on different domains.In the absence of 2m, most MHC1 molecules are not expressed efficiently on the surface of cells (12, 13). Although some MHC1 molecules, such as murine H-2L d and H-2D b , are transported to the cell surface without 2m, they have diminished levels of expression (14,15). This decreased MHC1 expression is not simply because of an export requirement for fully assembled MHC1 complexes. Transfection of 2m-negative cells with ER-retained 2m was able to salvage MHC1 cell surface expression (16). MHC1 folded in the presence of this ER-retained 2m was exported to the cell surface without bound 2m. Thus 2m, which promotes protein folding through a transient interaction, fits the definition of a chaperone (17). Therefore,...
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