title: Determination of the HLA-DM interaction site on HLA-DR molecules.
abstract: HLA-DM removes CLIP and other loosely bound peptides from MHC class II molecules. The crystal structures of class II molecules and of HLA-DM have not permitted identification of their interaction sites. Here, we describe mutations in class II that impair interactions with DM. Libraries of randomly mutagenized DR3 alpha and beta chains were screened for their ability to cause cell surface accumulation of CLIP/DR3 complexes in EBV-B cells. Seven mutations were associated with impaired peptide loading in vivo, as detected by SDS stability assays. In vitro, these mutant DR3 molecules were resistant to DM-catalyzed CLIP release and showed reduced binding to DM. All mutations localize to a single lateral face of HLA-DR, which we propose interacts with DM during peptide exchange.
authors.affiliation: School of Medicine, University of Pennsylvania, Philadelphia 19104, USA. rcdoebel@mail.med.upenn.edu
authors.forename: R C
authors.initials: RC
authors.lastname: Doebele
authors.forename: R
authors.initials: R
authors.lastname: Busch
authors.forename: H M
authors.initials: HM
authors.lastname: Scott
authors.forename: A
authors.initials: A
authors.lastname: Pashine
authors.forename: E D
authors.initials: ED
authors.lastname: Mellins
mesh_terms: D000595:Amino Acid Sequence; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells; D006681:HLA-D Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D006684:HLA-DR Antigens / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015803:HLA-DR3 Antigen / Q000235:genetics / Q000276:immunology; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007113:Immunity, Innate; D016130:Immunophenotyping; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; C416189:H2-M antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; D006684:HLA-DR Antigens; D015803:HLA-DR3 Antigen; D000949:Histocompatibility Antigens Class II; C045084:invariant chain; D012967:Sodium Dodecyl Sulfate