title: Avid interactions underlie the Lys63-linked polyubiquitin binding specificities observed for UBA domains.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Sims
authors.forename: Aydin
authors.initials: A
authors.lastname: Haririnia
authors.forename: Bryan C
authors.initials: BC
authors.lastname: Dickinson
authors.forename: David
authors.initials: D
authors.lastname: Fushman
authors.forename: Robert E
authors.initials: RE
authors.lastname: Cohen
mesh_terms: D001665:Binding Sites; D001667:Binding, Competitive; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D025821:Polyubiquitin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*
chemical_list: C510360:Ede1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D025821:Polyubiquitin; D005982:Glutathione Transferase; D008239:Lysine