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Updated: Jun 21, 2026

In Vitro Analysis of E3 Ubiquitin Ligase Function
Published on: May 14, 2021
Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains
Joshua J Sims1, Aydin Haririnia, Bryan C Dickinson
1Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
Abstract:
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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