Independent Interactions of Ubiquitin-Binding Domains in a Ubiquitin-Mediated Ternary Complex

Abstract

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.

Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

“…However, in contradiction to what would be expected if motions at distinct bonding sites were coupled, 54,55 a study of a ternary complex of ubiquitin failed to detect any allosteric effect between the hydrophobic patch and the β4-α2 loop interfaces. 56 Surprisingly, the picture of motions in ubiquitin derived from our chemical-exchange based study seems different from the one drawn from the analysis of RDCs. 25 This underlines the diversity of motions in ubiquitin as RDCs are more sensitive to fluctuations of the major conformer, while our work focuses on transitions to a weakly populated excited state.…”

Structure of the TbBILBO1 Protein N-terminal Domain from Trypanosoma brucei Reveals an Essential Requirement for a Conserved Surface Patch

“…Notable also is that the conserved surface patch is distal to this interaction surface. This could mean either that the function of the patch is unrelated to protein-protein interaction or that TbBILBO1 makes contacts via two separate interfaces as reported previously for regulating ubiquitin substrate release (25,26). Future work will test these hypotheses in depth.…”

Identification of a Non-covalent Ternary Complex Formed by PIAS1, SUMO1, and UBC9 Proteins Involved in Transcriptional Regulation

“…This result is equivalent with what has been recently found for the in vitro interaction between ubiquitin and either the ZNF216⅐p62 or ZNF4⅐UBE2D1 complexes. In the ZNF216⅐p62 and ZNF4⅐UBE2D1 complexes, the isolated A20 zinc-finger domain of either ZNF216 or ZNF4 contacts the ubiquitin polar patch, whereas the ubiquitin hydrophobic surface binds to either the UBA (ubiquitin-associated) domain of p62 or the backside of UBE2D1 (82,83). Thus, both SUMO and ubiquitin are highly versatile proteins that can function both as posttranslational modifying factors and as scaffolding proteins in non-covalent macromolecular assemblies.…”