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Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.


ABSTRACT: 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 DO?P11A (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 ?1 and ?2 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.

SUBMITTER: Yoon T 

PROVIDER: S-EPMC3396517 | biostudies-literature | 2012 Jul

REPOSITORIES: biostudies-literature

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Mapping the HLA-DO/HLA-DM complex by FRET and mutagenesis.

Yoon Taejin T   Macmillan Henriette H   Mortimer Sarah E SE   Jiang Wei W   Rinderknecht Cornelia H CH   Stern Lawrence J LJ   Mellins Elizabeth D ED  

Proceedings of the National Academy of Sciences of the United States of America 20120625 28


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 gener  ...[more]

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