Structural insights into the nanomolar affinity of RING E3 ligase ZNRF1 for Ube2N and its functional implications

Abstract

RING (Really Interesting New Gene) domains in ubiquitin RING E3 ligases exclusively engage ubiquitin (Ub)-loaded E2s to facilitate ubiquitination of their substrates. Despite such specificity, all RINGs characterized till date bind unloaded E2s with dissociation constants (Kds) in the micromolar to the sub-millimolar range. Here, we show that the RING domain of E3 ligase ZNRF1, an essential E3 ligase implicated in diverse cellular pathways, binds Ube2N with a Kd of ∼50 nM. This high-affinity interaction is exclusive for Ube2N as ZNRF1 interacts with Ube2D2 with a Kd of ∼1 µM, alike few other E3s. The crystal structure of ZNRF1 C-terminal domain in complex with Ube2N coupled with mutational analyses reveals the molecular basis of this unusual affinity. We further demonstrate that the ubiquitination efficiency of ZNRF1 : E2 pairs correlates with their affinity. Intriguingly, as a consequence of its high E2 affinity, an excess of ZNRF1 inhibits Ube2N-mediated ubiquitination at concentrations ≥500 nM instead of showing enhanced ubiquitination. This suggests a novel mode of activity regulation of E3 ligases and emphasizes the importance of E3-E2 balance for the optimum activity. Based on our results, we propose that overexpression-based functional analyses on E3 ligases such as ZNRF1 must be approached with caution as enhanced cellular levels might result in aberrant modification activity.

Structural basis of generic versus specific E2–RING E3 interactions in protein ubiquitination

“…in vitro data suggest that ZNRF1 might inhibit UBE2N activity via binding it tightly . The UBE2N:ZNRF1 complex structure illustrates the details of this astonishingly tight E2–E3 interaction and reveals that it is mediated by specialization of ZNRF1 within its canonical E2–E3 binding interface …”

“…(a) Ribbon representation of a single RNF4 monomer from UBE2D1~Ub:RNF4 complex (PDB: 4AP4) showing the zinc ions as spheres, the residues involved in zinc‐coordination and the allosteric linchpin residue as ball‐and‐sticks, and demarcating the E2‐binding site. The ribbon diagram is colored to reflect the average pairwise positional shift of each overlapping C α atom between RNF4 and unique RING cores from all available E2–RING E3 complex structures including RNF146 (PDB: 4QPL), TRIM25 (PDB: 5FER), BIRC2 (PDB: 6HPR), BIRC3 (PDB: 3 EB6), BIRC7 (PDB: 4AUQ), RNF38 (PDB: 4V3K), RNF25 (PDB: 5D1M), RNF165 (PDB: 5D0M), MDM2 (PDB: 5MNJ), TRIM23 (PDB: 5VZW), RNF13 (PDB: 5ZBU), E4B (PDB: 3L1Z), RNF2 (PDB: 3RPG), GP78 (PDB: 2LXP), SIZ1 (PDB: 5JNE), c‐CBL (PDB: 1FBV), RBX1 (PDB: 4P5O), TRAF6 (PDB: 3HCT), TRIM5 (PDB: 4TKP), RNF8 (PDB: 4WHV), ZNRF1 (PDB: 5YWR), LNX1 (PDB: 5H7S), CHIP (PDB: 2C2V), and FANCL (PDB: 2CCG) . (b) UBE2D1~Ub:RNF4 complex highlighting position of the RING domain relative to the E2, with the allosteric linchpin residue coordinating E2 as well as the donor Ub shown as ball‐and‐sticks.…”

“…Identification of the minimal set of interactions that are required for all E2–RING E3 interactions, which may be referred to as canonical E2–RING E3 interactions, can be achieved by comparison of the structures of the most versatile members of the E2 family. These are the closely related members of the UBE2D family, which build K48‐type Ub chains and target many regulatory proteins for destruction, and their distant relative UBE2N, which dimerise with catalytically‐dead E2 variants UBE2V1 or UBE2V2 to build K63‐type Ub chains and trigger immune responses . Remarkably, in vitro studies have shown that UBE2D1, UBE2D2, UBE2D3, and UBE2N can individually interact with over 30 RING E3s, with approximately 60% of these RING E3 interaction patterns overlapping .…”

“…The poorly characterized RING E3 ligase ZNRF1 was found to tightly interact with UBE2N in cells, a broad‐specificity E2 that associates with many other E3s to build K63‐type Ub chains . ZNRF1 is known to bind UBE2Ds and UBE2W, but it appears to be specialized to interact with UBE2N in particular, with its binding affinity against UBE2N being ~50 nM . in vitro data suggest that ZNRF1 might inhibit UBE2N activity via binding it tightly .…”

“…Specialized UBE2N‐ZNRF1 interaction facilitated by the RING domain itself. Ribbon representation of a single UBE2N:ZNRF1 dimer, with the second E2–RING E3 dimer hidden from view for clarity (PDB: 5YWR) . Zinc ions, residues involved in E2–RING E3 interactions and hydrogen‐bonding interactions are shown as spheres, ball‐and‐sticks and dashed lines, respectively.…”