Avid interactions underlie the K63-linked polyubiquitin binding specificities observed for UBA domains

Ubiquitin (Ub) receptor proteins as a group must contain a diverse set of binding specificities to distinguish the many forms of polyUb signals. Previous studies suggested that the large class of ubiquitin associated (UBA) domains contains members with intrinsic specificity for lysine 63-linked polyUb (K63-polyUb) or K48-polyUb, thus explaining how UBA-containing proteins can mediate diverse signaling events. Here we show that previously observed K63-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 K63-polyUb, but not K48-polyUb. Freed from GST, these UBAs are either non-selective or prefer K48-polyUb. Accordingly, NMR experiments reveal no K63-polyUb specific binding epitopes for these UBAs. We re-examine previous conclusions based on GST-UBAs and present an alternative model for how UBAs achieve a diverse range of linkage-specificities.