Thermodynamic and kinetic analysis of a peptide-class I MHC interaction highlights the noncovalent nature and conformational dynamics of the class I heterotrimer.

The class I major histocompatibility (MHC) molecule is a heterotrimer composed of a heavy chain, the small subunit beta(2)-microglobulin (beta(2)m), and a peptide. Fluorescence anisotropy has been used to assay the interaction of a labeled peptide with a recombinant, soluble form of the class I MHC HLA-A2. Consistent with earlier work, peptide binding is shown to be a two-step process limited by a conformational rearrangement in the heavy chain/beta(2)m heterodimer. However, we identify two pathways for peptide dissociation from the heterotrimer: (1) initial peptide dissociation leaving a heavy chain/beta(2)m heterodimer and (2) initial dissociation of beta(2)m, followed by peptide dissociation from the heavy chain. Eyring analyses of rate constants measured as a function of temperature permit for the first time a complete thermodynamic characterization of peptide binding. We find that in this case peptide binding is mostly entropically driven, likely reflecting the hydrophobic character of the peptide binding groove and the peptide anchor residues. Thermodynamic and kinetic analyses of peptide-MHC interactions as performed here may be of practical use in the engineering of peptides with desired binding properties and will aid in the interpretation of the effects of MHC and peptide substitutions on peptide binding and T cell reactivity. Finally, our data suggest a role for beta(2)m in dampening conformational dynamics in the heavy chain. Remaining conformational variability in the heavy chain once beta(2)m has bound may be a mechanism to promote promiscuity in peptide binding.