Ubiquitin (Ub) modifications are transduced by receptor proteins that use Ub-binding domains (UBDs) to recognize distinct interaction faces on the Ub surface. We report the nuclear magnetic resonance (NMR) solution structures of the A20-like zinc finger (A20 Znf) UBD of the Ub receptor ZNF216, and its complex with Ub, and show that the binding surface on Ub centered on Asp58 leaves the canonical hydrophobic Ile44 patch free to participate in additional interactions. We have modeled ternary complexes of the different families of UBDs and show that while many are expected to bind competitively to the same Ile44 surface or show steric incompatibility, other combinations (in particular, those involving the A20 Znf domain) are consistent with a single Ub moiety simultaneously participating in multiple interactions with different UBDs. We subsequently demonstrate by NMR that the A20 Znf domain of ZNF216 and the UBA domain of the p62 protein (an Ile44-binding UBD), which function in the same biological pathways, are able to form such a Ub-mediated ternary complex through independent interactions with a single Ub. This work supports an emerging concept of Ub acting as a scaffold to mediate multiprotein complex assembly.
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