A Point Mutation in the Groove of HLA-DO Allows Egress from the Endoplasmic Reticulum Independent of HLA-DM

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 102; no. 18; pp. 6443 - 6448
• Main Authors: Deshaies, Francis, Brunet, Alexandre, Diallo, Djibril A., Denzin, Lisa K., Samaan, Angela, Thibodeau, Jacques, Cresswell, Peter
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 03.05.2005; National Acad Sciences
• Subjects: Antibodies, Monoclonal - metabolism; Antigens; B lymphocytes; B-Lymphocytes - metabolism; Binding sites; Biological Sciences; Blotting, Western; Cell lines; Endoplasmic reticulum; Endoplasmic Reticulum - metabolism; Flow Cytometry; Fluorescence; HeLa Cells; Histograms; HLA-D Antigens - genetics; HLA-D Antigens - metabolism; Humans; Immunology; Immunoprecipitation; Lymphocyte receptors; Microscopy, Fluorescence; Molecular interactions; Molecules; Mutagenesis; Mutation; Peptides; Plasmids - genetics; Point Mutation - genetics; Protein Binding; Protein Structure, Tertiary; Protein Transport - physiology; Recombinant Fusion Proteins - metabolism; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0500853102

B lymphocytes express the nonclassical class II molecule HLA-DO, which modulates the peptide loading activity of HLA-DM in the endocytic pathway. Binding to HLA-DM is required for HLA-DO to egress from the endoplasmic reticulum (ER). To gain insights into the mode of action of DO and on the role of DM in ER release, we sought to identify DM-binding residues on DO. Our results show that DOα encompasses the binding site for HLA-DM. More specifically, mutation of residue DOα41 on an exposed lateral loop of the α1 domain affects the binding to DM, ER egress, and activity of DO. Using a series of chimeric DR/DO molecules, we confirmed the role of the α chain and established that a second DM-binding region is located C-terminal to the DOα80 residue, most probably in the α2 domain. Interestingly, after mutation of a buried proline (α11) on the floor of the putative peptide-binding groove, HLA-DO remained functional but became independent of HLA-DM for ER egress and intracellular trafficking. Collectively, these results suggest that the binding of HLA-DM to DOα allows the complex to egress from the ER by stabilizing intramolecular contacts between the N-terminal antiparallel β-strands of the DOαβ heterodimer.