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Assembly, analysis and architecture of atypical ubiquitin chains

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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Figure 1: NleL-mediated Ub-chain assembly.
Figure 2: Ub-chain restriction analysis using linkage-specific DUBs.
Figure 3: NleL assembles branched Ub chains.
Figure 4: Hydrolysis of branched and Lys6-linked polyUb by DUBs.
Figure 5: Large-scale assembly and crystal structures of Lys6-linked polyUb.
Figure 6: Solution studies of Lys6-linked diUb.
Figure 7: Solution studies of Lys6-linked triUb by NMR spectroscopy.
Figure 8: Ile36 patch-binding causes structural rearrangements in Ub.
Figure 9: Ub 'loop-in' and 'loop-out' conformations.

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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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Correspondence to David Komander.

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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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