Abstract
Ubiquitin (Ub) chains regulate many cellular processes, but several chain types including Lys6 linkages have remained unstudied. Here we analyze the bacterial effector E3 ligase NleL (non-Lee–encoded effector ligase) from enterohemorrhagic Escherichia coli (EHEC) O157:H7, which assembles Lys6- and Lys48-linked Ub polymers. Using linkage-specific human deubiquitinases (DUBs) we show that NleL generates heterotypic Ub chains, and branched chains are efficiently hydrolyzed by DUBs. USP family DUBs cleave Lys6-linked polymers exclusively from the distal end, whereas a DUB with preference for Lys6 can cleave Lys6-linked polymers at any position in the chain. We used NleL to generate large quantities of Lys6-linked polyUb. Crystallographic and NMR spectroscopy analyses revealed that an asymmetric interface between Ile44 and Ile36 hydrophobic patches of neighboring Ub moieties is propagated in longer Lys6-linked Ub chains. Interactions via the Ile36 patch can displace Leu8 from the Ile44 patch, leading to marked structural perturbations of Ub.
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Acknowledgements
We thank D.Y. Lin and J. Chen (Purdue University) for providing the NleL plasmid, and T. Rutherford and members of the Komander laboratory for helpful discussions. We acknowledge T. Mevissen, D. Tourigny and T. Wauer for insights into combinatorial mathematics. Crystallographic data were collected at the European Synchrotron Radiation Facility at beam lines ID23-1, ID29 and ID14-4. This work was supported by the Medical Research Council (U105192732) and the European Molecular Biology Organization Young Investigator Programme.
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M.K.H. and D.K. designed, and M.K.H. performed and analyzed all experiments in this manuscript, with help from S.M.V.F. for NMR analysis. D.K. wrote the manuscript with help from all authors.
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D.K. is a consultant for Mission Therapeutics.
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Hospenthal, M., Freund, S. & Komander, D. Assembly, analysis and architecture of atypical ubiquitin chains. Nat Struct Mol Biol 20, 555–565 (2013). https://doi.org/10.1038/nsmb.2547
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DOI: https://doi.org/10.1038/nsmb.2547
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