Dynamics of peptide binding to purified antibody-bound H-2Db and H-2Db beta 2m complexes.
Although it is clear that each component of the class I MHC trimolecular complex (heavy chain, beta 2m, and antigenic peptide) contributes to its formation and stability, the specific interaction governing assembly and disassembly remain to be clarified. In an effort to address these issues using purified H-2Db molecules, we used a solid-phase binding assay recently developed in our laboratory to quantify kinetic parameters for class I assembly and disassembly. It was found that the influenza NP peptide Y367-374 associated with preformed empty complexes of 28-14-8S- (i.e., anti-alpha 3) bound Db beta 2m dimers much more quickly (t 1/2 < 0.2 h at 22 degrees C) than it did when coincubated with an anti-alpha 3 bound Db and human beta 2m (t1/2 3.5 h at 22 degrees C). The previously reported potential for the NP peptide Y367-374 to interact directly with free Db heavy chains and configure the conventionally beta 2m-dependent B22 epitope in the absence of beta 2m, was confirmed using our assay system. However, the rate of B22 epitope formation induced in the Db heavy chain by NP Y367-374 was considerably slower (t1/2 13 h, at 22 degrees C) and much less efficient on a molar basis than that resulting from the addition of beta2m (t1/2, 0.75 h, at 22 degrees C). In contrast, the Db heavy chain with NP-Y367-374 was more resistant to thermal disassembly (as measured by loss of the B22 epitope, t1/2 2h, 37 degrees C) than the Db beta 2m empty dimer (t1/2 0.2 h). Finally, stability of the preformed trimolecular complex of heavy chain, beta 2m, and peptide was found to diminish in accordance with deviation of the peptide from the optimal length and with increasing temperature from 4 to 37 degrees C.
Burshtyn
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Department of Immunology, University of Toronto, Canada.
Barber
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D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes; D006183:H-2 Antigens / Q000378:metabolism*; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000276:immunology; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D013696:Temperature; D014758:Viral Core Proteins / Q000276:immunology / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000276:immunology*
D000956:Antigens, Viral; D000939:Epitopes; D006183:H-2 Antigens; D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010455:Peptides; D014758:Viral Core Proteins; D001613:beta 2-Microglobulin