title: Analysis of two acidic P6 pocket residues in the pH dependency of peptide binding by I-E(k).
abstract: Peptide binding to major histocompatibility complex (MHC) class II molecules is optimal at mildly acidic pH. X-ray crystal structures solved for the murine class II molecule I-E(k) revealed an interesting localization of negatively charged residues within the P6 pocket, which may have implications in the pH dependency of peptide binding. Protonation of these critical residues, under acidic conditions, has been proposed to be important for the formation of stable class II-peptide complexes. In this study, we address a possible role for these charged residues in the pH dependency of peptide binding. An I-E(k) mutant was generated in which two acidic residues of the P6 pocket were substituted with uncharged residues. This class II mutant was expressed, purified, and tested for its ability to bind peptides. The mutant I-E(k) was observed to load peptides optimally at mildly acidic pH. Peptide binding to the mutant was enhanced in the presence of DM, and optimal DM-enhanced binding occurred in the acidic pH range. These findings indicate that structural changes other than protonation of acidic residues in pocket 6 must play a dominant role in pH-regulated peptide binding to I-E(k).
authors.affiliation: Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA.
authors.forename: Chinh T
authors.initials: CT
authors.lastname: Dao
authors.forename: Dominique A
authors.initials: DA
authors.lastname: Weber
authors.forename: Peter E
authors.initials: PE
authors.lastname: Jensen
mesh_terms: D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006681:HLA-D Antigens / Q000378:metabolism; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009154:Mutation; D010455:Peptides / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D006681:HLA-D Antigens; C084245:HLA-DM antigens; D000949:Histocompatibility Antigens Class II; C024138:I-E-antigen; D010455:Peptides; D001224:Aspartic Acid; D018698:Glutamic Acid