Designing active RNF4 monomers by introducing a tryptophan: avidity towards E2∼Ub conjugates dictates the activity of ubiquitin RING E3 ligases.

Ubiquitin RING E3 ligases (E3s) catalyze ubiquitin (Ub) transfer to their substrates by engaging E2∼Ub intermediates with the help of their RING domains. Different E3s have been found to contain a conserved tryptophan residue in their RING that plays an essential role in E2 binding and, hence, enzymatic activity. Many active E3s, however, lack this specific residue. We mined through the existing data to observe that the conservation of the tryptophan and quaternary organization of the RING domains are remarkably correlated. Monomeric RINGs possess the tryptophan while all well-characterized dimeric RINGs, except RNF8, contain other amino acid residues. Biochemical analyses on representative E3s and their mutants reveal that the tryptophan is essential for optimal enzymatic activity of monomeric RINGs whereas dimeric E3s with tryptophan display hyperactivity. Most critically, the introduction of the tryptophan restores the activity of inactive monomeric RNF4 mutants, an obligatory dimeric E3. Binding studies indicate that monomeric RINGs retained the tryptophan for their optimal functionality to compensate for weak Ub binding. On the other hand, tryptophan was omitted from dimeric RINGs during the course of evolution to prevent unwanted modifications and allow regulation of their activity through oligomerization.

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

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.

RING E3-Catalyzed E2 Self-Ubiquitination Attenuates the Activity of Ube2E Ubiquitin-Conjugating Enzymes.

Ubiquitination of a target protein is accomplished through sequential actions of the E1, E2s, and the E3s. E2s dictate the modification topology while E3 ligases confer substrate specificity and recruit the cognate E2. Human genome codes for ~35 different E2 proteins; all of which contain the characteristic ubiquitin-conjugating UBC core domain sufficient for catalysis. Many of these E2 enzymes also have N- or C-terminal extensions; roles of which are not very well understood. We show that the N-terminal extension of Ube2E1 undergoes intramolecular auto-ubiquitination. This self-ubiquitination activity is enhanced in the presence of interacting RING E3 ligases and results in a progressive attenuation of the E2 activity toward substrate/E3 modification. We also find that the N-terminal ubiquitination sites are conserved in all the three Ube2Es and replacing them with arginine renders all three full-length Ube2Es equally active as their core UBC domains. Based on these results, we propose that E3-catalyzed self-ubiquitination acts as a key regulatory mechanism that controls the activity of Ube2E class of ubiquitin E2s.