The beta 2-microglobulin dissociation rate is an accurate measure of the stability of MHC class I heterotrimers and depends on which peptide is bound.
Stable, recombinant, water-soluble complexes of HLA-A2 and HLA-B27 were reconstituted from 125I-labeled beta 2-microglobulin (beta 2m), a synthetic peptide, and HLA H chain fragments expressed as inclusion bodies in the Escherichia coli cytoplasm. Using this system, we were able to show: 1) the t1/2 of beta 2m dissociation from HLA complexes at 37 degrees C varied from approximately 40 h to less than 1 h, depending on the peptide employed for reconstitution. Peptide length and composition were found to be critical factors in determining the beta 2m dissociation rate. Endogenous peptides form complexes that are about as stable as those formed with typical antigenic peptides. 2) Peptide exchange reactions, in which an exogenous peptide replaces the peptide that is already bound by the class I molecule, proceed readily for complexes that have rapid beta 2m dissociation rates. Thus, difficulties in demonstrating peptide binding to complexes that contain endogenous peptides can be attributed to the stability of the endogenous peptide/class I molecule complex. 3) The peptide exchange reaction does not require concomitant beta 2m dissociation. 4) Distal parts of the class I molecule, which are not directly involved in peptide binding or beta 2m binding, have a major impact on the stability of class I molecules. Thus, these studies show that the dissociation rate of beta 2m is an excellent measure of how tightly a given peptide binds to class I MHC molecules, that the ability to bind peptide is tightly coupled to the binding of beta 2m and vice versa, and that regions of the molecule distal from the binding site influence the stability of peptide binding.
Parker
K C
KC
Biological Resources Branch, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
DiBrino
M
M
Hull
L
L
Coligan
J E
JE
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry; D015395:Histocompatibility Antigens Class I / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry / Q000276:immunology; D001613:beta 2-Microglobulin / Q000737:chemistry*
D000956:Antigens, Viral; D015395:Histocompatibility Antigens Class I; D046911:Macromolecular Substances; D010455:Peptides; D014764:Viral Proteins; D001613:beta 2-Microglobulin