pH Affects both the mechanism and the specificity of peptide binding to a class II major histocompatibility complex molecule

Abstract

We have compared the contribution of electrostatic forces in the binding of antigenic peptides to the class II MHC molecule, IEk, at weakly acidic (pH 5.4) and neutral (pH 7.5) pH values. The binding of specific moth cytochrome c (MCC) and hemoglobin (Hb) peptides to IEk is very sensitive to ionic strength at pH 7.5 but not at pH 5.4, indicating 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 least 30-fold, implicating this residue in the pH-dependent electrostatic interaction. The presence of a C-terminal Lys in many of the peptides that are restricted to IEk suggests that this electrostatic interaction is widely used to bind peptides to this MHC molecule. We also probed the electrostatic environment of the peptide binding groove adjacent to the N-terminus of the bound peptide by rapid-diffusion fluorescence energy transfer using a terbium-labeled MCC peptide. In this region 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 carboxylates to the mechanism and specificity of peptide binding. The biological importance of having two distinct mechanisms of peptide binding at different pH may be that it acts to broaden the spectrum of antigenic peptides that can be presented to T-cells.

Stability of empty and peptide-loaded class II major histocompatibility complex molecules at neutral and endosomal pH: Comparison to class I proteins

“…Apparently, these GPI-linked chimeras do not traffic through the cell's endosomal compartments and consequently lack bound peptides as evidenced by labeled peptide binding experiments (19), by the fact that no peptides can be eluted from the molecules (H. Schild, P. A. Reay, and M.M.D., unpublished results), by the observation that the I-E k bearing cells were unable to present endogenously processed peptides to a specific T-cell line (18), and by their distinct unfolding behavior and stability as described herein. I-E k molecules complexed with moth cytochrome c (residues 88-103) peptide were prepared and purified as reported (26,27). Soluble I-A d (mouse) with E␣ peptide (residues 52-68) covalently linked to the heterodimer ␤ chain was constructed by exchanging the peptide portion of I-A d ͞OVA cDNA (28) by that of the E␣ sequence obtained from an I-A b ͞E␣ construct (29).…”

T-cell triggering thresholds are modulated by the number of antigen within individual T-cell receptor clusters

“…Therefore we expect uncorrelated binding of both peptide-MHC complexes. The peptide off rate is extremely slow and peptide reloading from solution is negligible at neutral pH (27). Thus the original loading configuration is expected to persist throughout the entire experiment, which results in a Poisson distribution for the number of agonist MCC peptides in each grid box.…”

The accessibility of peptides bound to the mouse MHC class II molecule IEd studied by fluorescence

“…groove of IEd is lined with negatively charged amino acid residues [ 161, this reduced quenching may be due to electrostatic repulsion of the anionic I- [17]. Therefore the experiment was repeated using the uncharged nitroxide radical TEMPOL, which quenches fluorescein fluorescence very efficiently by a mechanism involving electron exchange [18].…”

“…The data point to a structural model in which the N-termini of HEL and dyn are buried in, rather than protruding from, the IEd binding groove. In this respect the IEd-peptide complexes studied here, differ from the complex of IEk with the moth cyto-233 chrome c 88-103 peptide where the N-terminus was shown to extend out of the binding groove [9,17]. Given the ability of the class II binding groove to accomodate a stretch of approximately 15 amino acid residues [ 11, the shorter lengths of HEL and dyn compared to the cytochrome c peptide may account for this difference.…”