Structural and functional studies of major histocompatibility complex class II

Antigen presentation mediated by major histocompatibility complex class II molecules is important in the defense against extracellular organisms. Pathogens which are phagocytosed and endocytosed are degraded in endosomal and lysosomal compartments where MHC class II molecules gain access to the processed antigens. Multiple elements of the class II antigen processing and presentation pathway help ensure and optimize peptide loading in these compartments. The mildly acidic pH environment of the endosomes and lysosomes promotes peptide binding in several ways. Structural transitions induced in the class II molecules by low pH are associated with enhanced peptide binding. The crystal structures solved for the murine MHC class II, I-Ek, revealed an interesting localization of negatively charged residues which had possible implications to I-Ek pH-dependent peptide binding. The importance of this structural feature was addressed by making a mutant I-Ek molecule lacking the cluster of acidic residues. We observed little difference in the pH optima of peptide binding between mutant and wild-type I-Ek molecules. Moreover, structural transitions seen in both mutant and wild-type molecules were identical. In addition, peptide exchange was catalyzed by HLA-DM from the mutant I-Ek despite the P6 pocket mutations.; The endosomal resident non-classical class II molecule, HLA-DM, exchanges peptides from MHC class II molecule at mildly acidic pH. The mechanism of its catalysis has not yet been completed, but many details have been uncovered. Several groups have hypothesized that a network of hydrogen bonds formed between conserved MHC class II residues and the carbon backbone of the peptide may be an important structure in the catalysis of peptide exchange. To test this hypothesis, we generated an extensive set of DR1 hydrogen bond mutants, making alanine substitutions to selected DR1 residues. DM function was tested on all DR1 mutants, and in all cases, DM was able to catalyze peptide release. However, when the potency of DM activity was closely examined, we observed that a set of DR1 mutants possessed depressed HLA-DM potency in comparison to the wild-type. Potency of DM catalysis was represented as a ratio between DM-mediated peptide exchange rates and intrinsic complex stability. The results underscore the importance of a set of conserved class II residues and their H-bonds to complex stability and may suggest a role for class II hydrogen bond sets in the mechanism of DM-catalyzed peptide exchange.