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Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase


Ubiquitination is a reversible post-translational modification that regulates a myriad of eukaryotic functions. Our ability to study the effects of ubiquitination is often limited by the inaccessibility of homogeneously ubiquitinated proteins. In particular, elucidating the roles of the so-called 'atypical' ubiquitin chains (chains other than Lys48- or Lys63-linked ubiquitin), which account for a large fraction of ubiquitin polymers, is challenging because the enzymes for their biosynthesis are unknown. Here we combine genetic code expansion, intein chemistry and chemoselective ligations to synthesize 'atypical' ubiquitin chains. We solve the crystal structure of Lys6-linked diubiquitin, which is distinct from that of structurally characterized ubiquitin chains, providing a molecular basis for the different biological functions this linkage may regulate. Moreover, we profile a panel containing 10% of the known human deubiquitinases on Lys6- and Lys29-linked ubiquitin and discover that TRABID cleaves the Lys29 linkage 40-fold more efficiently than the Lys63 linkage.

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Figure 1: Synthesis and characterization of Lys6- and Lys29-linked ubiquitin.
Figure 2: Structure of Lys6-linked diubiquitin.
Figure 3: Profiling of deubiquitinase activity toward (UbLys6)2, (UbLys29)2 and (UbLys63)2.
Figure 4: The specificity constant of TRABID is 40-fold higher on (UbLys29)2 than on (UbLys63)2 as determined by quantitative western blot.

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We thank K.D. Wilkinson (Emory University), B. Kessler (Oxford University), S. Urbe, M. Clague (Liverpool University) and P. Cohen (MRC Protein Phosphorylation Unit, Dundee) for constructs, S. Peak-Chew, F. Begum and E. Stephens for MS/MS and members of the Laboratory of Molecular Biology for reagents. We thank L. James (Laboratory of Molecular Biology) for suggestions on kinetic analysis. This work was supported by the Medical Research Council, United Kingdom.

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Authors and Affiliations



J.W.C. and D.K. initiated the study. J.W.C. and S.V. designed the ubiquitin chain synthesis and developed the quantitative blot method for deubiquitinase kinetics. S.V. implemented and characterized the synthesis with assistance from D.P.N. and implemented and analyzed the quantitative kinetics on TRABID by western blot. S.V. and D.K. performed structural studies, analyzed structural data and designed and analyzed the deubiquitinase screen. Y.Y. and D.K. developed the fluorescent deubiquitinase assay that S.V. applied to estimate the Kms for TRABID. S.V., D.K. and J.W.C. analyzed the data and wrote the paper.

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Correspondence to David Komander or Jason W Chin.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods (PDF 2309 kb)

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Virdee, S., Ye, Y., Nguyen, D. et al. Engineered diubiquitin synthesis reveals Lys29-isopeptide specificity of an OTU deubiquitinase. Nat Chem Biol 6, 750–757 (2010).

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