title: Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove.
abstract: Heat-shock protein 70 (Hsp70)-peptide complexes are involved in MHC class I- and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70-HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously.
authors.affiliation: Children's Hospital, University of Tübingen, Tübingen, Germany.
authors.forename: Karin M
authors.initials: KM
authors.lastname: Rohrer
authors.forename: Markus
authors.initials: M
authors.lastname: Haug
authors.forename: Daniela
authors.initials: D
authors.lastname: Schwörer
authors.forename: Hubert
authors.initials: H
authors.lastname: Kalbacher
authors.forename: Ursula
authors.initials: U
authors.lastname: Holzer
mesh_terms: D001665:Binding Sites / Q000235:genetics; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology; D049109:Cell Proliferation; D002478:Cells, Cultured; D006684:HLA-DR Antigens / Q000276:immunology* / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D010455:Peptides / Q000276:immunology*; D013379:Substrate Specificity
chemical_list: D006684:HLA-DR Antigens; D018840:HSP70 Heat-Shock Proteins; D010455:Peptides
keywords: CD4+ T-cell proliferation; HLA-DR binding; dissociation-enhanced lanthanide fluorescent immunoassay; heat-shock proteins 70; point mutations