Abstract
Class I major histocompatibility complexes (MHC) are heterotrimeric structures comprising heavy chains (HC), beta2-microglobulin (beta2-m), and short antigenic peptides of 8-10 amino acids. These components assemble in the endoplasmic reticulum and are released to the cell surface only when a peptide of the appropriate length and sequence is incorporated into the structure. The binding of beta2-m and peptide to HC is cooperative, and there is indirect evidence that the formation of a stable heterotrimer from an unstable HC:beta2-m heterodimer involves a peptide-induced conformational change in the HC. Such a conformational change could ensure both a strong interaction between the three components and also signal the release of stably assembled class I MHC molecules from the endoplasmic reticulum. A peptide-induced conformational change in HC has been demonstrated in cell lysates lacking beta2-m to which synthetic peptides were added. Many features of this conformational change suggest that it may be physiologically relevant. In an attempt to study the peptide-induced conformational change in detail we have expressed a soluble, truncated form of the mouse H-2Db HC that contains only the peptide binding domains of the class I molecule. We have shown that this peptide-binding "platform" is relatively stable in physiological buffers and undergoes a conformational change that is detectable with antibodies, in response to synthetic peptides. We also show that the structural features of peptides that induce this conformational change in the platform are the same as those required to observe the conformational change in full-length HC. In this respect, therefore, the HC alpha1 and alpha2 domains, which together form the peptide binding site of class I MHC, are able to act independently of the rest of the molecule.
Highlights
Glycoproteins encoded by the major histocompatibility complex (MHC)1 class I locus present peptide antigens to cytotoxic T lymphocytes
In the absence of bound 2-m, free heavy chains (HC) undergoes a conformational change that is detectable with antibodies (4, 14 – 16), and similarities in the structural requirements for this conformational change and those for stable peptide binding to HC:2-m heterodimers suggest that this conformational change may be related or identical to that proposed for the HC:2-m complex upon peptide binding [12, 17]
There is a more stringent requirement for both peptide anchor residues for the incorporation of peptide into refolded Db in the absence of 2-m. This is the first description of a soluble fragment of an MHC class I molecule comprising the two N-terminal domains of the protein that form the antigenic peptide-binding site
Summary
Cells, and Antibodies—pGMT7 is a 3.1-kilobase pair, pAR2067-based plasmid containing T7 promoter and terminators flanking a polylinker. E. coli strain BL21 DE(plys c)is amp incorporates an isopropyl-1-thio--D-galactopyranoside-inducible gene for T7 RNA polymerase, allowing high level, inducible expression of transfected DNA. The monoclonal antibodies B22.249 and 27-11-13 [4, 18] recognize the native conformation of the H2-Db heavy chain. It recognizes epitopes on both Db and Kb heavy chains, which are lost upon 2-m binding but remain when heavy chains bind to peptides in the absence of 2-m
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