Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.
HLA-DO (DO) is a nonclassic class II heterodimer that inhibits the action of the class II peptide exchange catalyst, HLA-DM (DM), and influences DM localization within late endosomes and exosomes. In addition, DM acts as a chaperone for DO and is required for its egress from the endoplasmic reticulum (ER). These reciprocal functions are based on direct DO/DM binding, but the topology of DO/DM complexes is not known, in part, because of technical limitations stemming from DO instability. We generated two variants of recombinant soluble DO with increased stability [zippered DOαP11A (szDOv) and chimeric sDO-Fc] and confirmed their conformational integrity and ability to inhibit DM. Notably, we found that our constructs, as well as wild-type sDO, are inhibitory in the full pH range where DM is active (4.7 to ∼6.0). To probe the nature of DO/DM complexes, we used intermolecular fluorescence resonance energy transfer (FRET) and mutagenesis and identified a lateral surface spanning the α1 and α2 domains of szDO as the apparent binding site for sDM. We also analyzed several sDM mutants for binding to szDOv and susceptibility to DO inhibition. Results of these assays identified a region of DM important for interaction with DO. Collectively, our data define a putative binding surface and an overall orientation of the szDOv/sDM complex and have implications for the mechanism of DO inhibition of DM.
Department of Pediatrics, Program in Immunology, Stanford University, Stanford, CA 94305, USA.
Taejin
T
Yoon
Henriette
H
Macmillan
Sarah E
SE
Mortimer
Wei
W
Jiang
Cornelia H
CH
Rinderknecht
Lawrence J
LJ
Stern
Elizabeth D
ED
Mellins
D000818:Animals; D017951:Antigen Presentation; D000941:Antigens / Q000737:chemistry; D001665:Binding Sites; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006681:HLA-D Antigens / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008958:Models, Molecular; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D055672:Static Electricity
D000941:Antigens; D006681:HLA-D Antigens; C084245:HLA-DM antigens; C046148:HLA-DO antigens; D010455:Peptides; D011134:Polysaccharides; D011994:Recombinant Proteins