Ubiquitin (denoted Ub) receptor proteins as a group must contain a diverse set of binding specificities to distinguish the many forms of polyubiquitin (polyUb) signals. Previous studies suggested that the large class of ubiquitin-associated (UBA) domains contains members with intrinsic specificity for Lys63-linked polyUb or Lys48-linked polyUb, thus explaining how UBA-containing proteins can mediate diverse signaling events. Here we show that previously observed Lys63-polyUb selectivity in UBA domains is the result of an artifact in which the dimeric fusion partner, glutathione S-transferase (GST), positions two UBAs for higher affinity, avid interactions with Lys63-polyUb, but not with Lys48-polyUb. Freed from GST, these UBAs are either nonselective or prefer Lys48-polyUb. Accordingly, NMR experiments reveal no Lys63-polyUb–specific binding epitopes for these UBAs. We reexamine previous conclusions based on GST-UBAs and present an alternative model for how UBAs achieve a diverse range of linkage specificities.
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Scientific Reports Open Access 07 December 2020
Structural insights into two distinct binding modules for Lys63-linked polyubiquitin chains in RNF168
Nature Communications Open Access 12 January 2018
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We thank P. Cole (Johns Hopkins School of Medicine) for materials and advice for fluorescent labeling by expressed protein ligation. Work in this study was supported in part by the US National Istitutes of Health (NIH) grant GM065334 (to D.F.) and NIH Roadmap grant RR020839.
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Sims, J., Haririnia, A., Dickinson, B. et al. Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains. Nat Struct Mol Biol 16, 883–889 (2009). https://doi.org/10.1038/nsmb.1637
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