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HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism

Nature Structural & Molecular Biology volume 20, pages 90–98 (2013)Cite this article

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Abstract

Mammalian class II major histocompatibility (MHCII) proteins bind peptide antigens in endosomal compartments of antigen-presenting cells. The nonclassical MHCII protein HLA-DM chaperones peptide-free MHCII, protecting it against inactivation, and catalyzes peptide exchange on loaded MHCII. Another nonclassical MHCII protein, HLA-DO, binds HLA-DM and influences the repertoire of peptides presented by MHCII proteins. However, the mechanism by which HLA-DO functions is unclear. Here we have used X-ray crystallography, enzyme kinetics and mutagenesis approaches to investigate human HLA-DO structure and function. In complex with HLA-DM, HLA-DO adopts a classical MHCII structure, with alterations near the α subunit's 310 helix. HLA-DO binds to HLA-DM at the same sites implicated in MHCII interaction, and kinetic analysis showed that HLA-DO acts as a competitive inhibitor. These results show that HLA-DO inhibits HLA-DM function by acting as a substrate mimic, and the findings also limit the possible functional roles for HLA-DO in antigen presentation.

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Figure 1: HLA-DO binds to HLA-DM and inhibits DM-catalyzed peptide binding and release.
Figure 2: Structure of the DO–DM complex.
Figure 3: The HLA-DO structure is very similar to conventional MHCII proteins, but with differences in the α subunit's 310 helix and adjacent extended strand.
Figure 4: Mutations that alter DM-MHCII interaction map to the DO–DM interface.
Figure 5: DM mutations at the DO interface have similar effects on interactions with DR and DO.
Figure 6: Kinetic analysis of DO inhibition of DM-catalyzed peptide exchange.

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Acknowledgements

We thank L. Karlsson (Johnson & Johnson) for S2 cells expressing DM and DO-Fc, J. Thibodeau (Université de Montréal) for plasmid pRmHa3 encoding sDOα and sDOβ cDNAs, L. Lee and L. Lu for assistance with insect cell culture and protein purification, H. Robinson and A. Heroux for assistance with crystallographic data collection, and E. Stratikos for helpful comments. Data for this study were measured at beamlines X25 and X29 of the US National Synchrotron Light Source, with support from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the US Department of Energy and from the National Center for Research Resources (NCRR) of the US National Institutes of Health (NIH). This work was supported by NIH grants AI-38996 (L.J.S.), AI-48833 (L.J.S.), T32 AI07349 (S.E.M.), F32 AI072984 (S.E.M.), AI-095813 (E.D.M.) and AI-075253 (E.D.M.), by NIH/NCRR Clinical and Translational Science Award UL1 RR025744 at Stanford University, and by the Lucile Packard Foundation for Children's Health (E.D.M.).

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  1. Taejin Yoon

    Present address: Current address: Department of Chemistry, Yonsei University, Seoul, Korea.,

  2. Abigail I Guce and Sarah E Mortimer: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Abigail I Guce, Sarah E Mortimer, Corrie A Painter & Lawrence J Stern

  2. Department of Pediatrics, Stanford University, Stanford, California, USA

    Taejin Yoon, Wei Jiang & Elizabeth D Mellins

  3. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts, USA

    Lawrence J Stern

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Contributions

S.E.M. prepared and crystallized the DO–DM complex; S.E.M., A.I.G. and L.J.S. interpreted diffraction data; A.I.G. built the structural model; S.E.M. performed kinetics experiments; T.Y., W.J. and A.I.G. analyzed mutant proteins; A.I.G., S.E.M., T.Y., C.A.P., W.J., E.D.M. and L.J.S. designed experiments and analyzed data; A.I.G., S.E.M., C.A.P., W.J., E.D.M. and L.J.S. wrote the manuscript.

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Correspondence to Lawrence J Stern.

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Guce, A., Mortimer, S., Yoon, T. et al. HLA-DO acts as a substrate mimic to inhibit HLA-DM by a competitive mechanism. Nat Struct Mol Biol 20, 90–98 (2013). https://doi.org/10.1038/nsmb.2460

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