Molecular Cell
Volume 68, Issue 1, 5 October 2017, Pages 233-246.e5
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Ubiquitin Linkage-Specific Affimers Reveal Insights into K6-Linked Ubiquitin Signaling

https://doi.org/10.1016/j.molcel.2017.08.020Get rights and content
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Highlights

  • Respective linkage-specific affimers recognize K6- or K33-/K11-linked chains

  • Structures of affimer:diUb complexes reveal mechanisms of linkage specificity

  • Improved affimers can be used in western blotting, confocal microscopy, and pull-downs

  • Pull-downs with K6-specific affimers reveal HUWE1 to be a major K6 ligase in cells

Summary

Several ubiquitin chain types have remained unstudied, mainly because tools and techniques to detect these posttranslational modifications are scarce. Linkage-specific antibodies have shaped our understanding of the roles and dynamics of polyubiquitin signals but are available for only five out of eight linkage types. We here characterize K6- and K33-linkage-specific “affimer” reagents as high-affinity ubiquitin interactors. Crystal structures of affimers bound to their cognate chain types reveal mechanisms of specificity and a K11 cross-reactivity in the K33 affimer. Structure-guided improvements yield superior affinity reagents suitable for western blotting, confocal fluorescence microscopy and pull-down applications. This allowed us to identify RNF144A and RNF144B as E3 ligases that assemble K6-, K11-, and K48-linked polyubiquitin in vitro. A protocol to enrich K6-ubiquitinated proteins from cells identifies HUWE1 as a main E3 ligase for this chain type, and we show that mitofusin-2 is modified with K6-linked polyubiquitin in a HUWE1-dependent manner.

Keywords

Lys6-linked ubiquitin chains
affimer
mitophagy
Parkin
HUWE1
Mfn2
X-ray crystallography
microscale thermophoresis

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Present address: Institute of Structural and Molecular Biology, University College London and Birkbeck, Malet Street, London WC1E 7HX, UK

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