PH AFFECTS BOTH THE MECHANISM AND THE SPECIFICITY OF PEPTIDE BINDING TO A CLASS-II MAJOR HISTOCOMPATIBILITY COMPLEX MOLECULE

We have compared the contribution of electrostatic forces in the bindi ng of antigenic peptides to the class II MHC molecule, IE(k), at weakl y acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of speci fic moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IE(k) is v ery sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicatin g that the mechanism of peptide binding is pH-dependent. Substitution of the C-terminal Lys in MCC for an Ala residue selectively destroyed peptide binding at neutral pH and increased the dissociation rate at l east 30-fold, implicating this residue in the pH-dependent electrostat ic interaction. The presence of a C-terminal Lys in many of the peptid es that are restricted to IE(k) suggests that this electrostatic inter action is widely used to bind peptides to this MHC molecule. We also p robed the electrostatic environment of the peptide binding groove adja cent to the N-terminus of the bound peptide by rapid-diffusion fluores cence energy transfer using a terbium-labeled MCC peptide. In this reg ion of the peptide binding groove, more negative charge is present at pH 7.5 than at pH 5.4. These findings indicate the importance of MHC c arboxylates to the mechanism and specificity of peptide binding. The b iological importance of having two distinct mechanisms of peptide bind ing at different pH may be that it acts to broaden the spectrum of ant igenic peptides that can be presented to T-cells.