scholarly journals RING domain dimerization is essential for RNF4 function

2010 ◽  
Vol 431 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Chu Wai Liew ◽  
Huaiyu Sun ◽  
Tony Hunter ◽  
Catherine L. Day
Keyword(s):  
Ubiquitin Ligases ◽  
Ring Domain ◽  
E3 Ligases ◽  
Finger Protein ◽  
Thioester Bond ◽  
New Gene ◽  
The Stability ◽  
Ring E3 Ligases ◽  
Ring E3

RNF4 [RING (really interesting new gene) finger protein 4] family ubiquitin ligases are RING E3 ligases that regulate the homoeostasis of SUMOylated proteins by promoting their ubiquitylation. In the present paper we report that the RING domain of RNF4 forms a stable dimer, and that dimerization is required for ubiquitin transfer. Our results suggest that the stability of the E2~ubiquitin thioester bond is regulated by RING domain dimerization.

Regulation of ubiquitin transfer by XIAP, a dimeric RING E3 ligase

2013 ◽  
Vol 450 (3) ◽  
pp. 629-638 ◽  
Author(s):  
Yoshio Nakatani ◽  
Torsten Kleffmann ◽  
Katrin Linke ◽  
Stephen M. Condon ◽  
Mark G. Hinds ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
E3 Ligase ◽  
Ring Domain ◽  
Aromatic Residue ◽  
E3 Ligases ◽  
Dimer Interface ◽  
Ring E3 Ligase ◽  
Thioester Bond ◽  
Ring E3 ◽  
Apoptosis Proteins

RING domains of E3 ligases promote transfer of Ub (ubiquitin) from the E2~Ub conjugate to target proteins. In many cases interaction of the E2~Ub conjugate with the RING domain requires its prior dimerization. Using cross-linking experiments we show that E2 conjugated ubiquitin contacts the RING homodimer interface of the IAP (inhibitor of apoptosis) proteins, XIAP (X-linked IAP) and cIAP (cellular IAP) 2. Structural and biochemical analysis of the XIAP RING dimer shows that an aromatic residue at the dimer interface is required for E2~Ub binding and Ub transfer. Mutation of the aromatic residue abolishes Ub transfer, but not interaction with Ub. This indicates that nuleophilic attack on the thioester bond depends on precise contacts between Ub and the RING domain. RING dimerization is a critical activating step for the cIAP proteins; however, our analysis shows that the RING domain of XIAP forms a stable dimer and its E3 ligase activity does not require an activation step.

scholarly journals The deubiquitylase USP9X controls ribosomal stalling

2020 ◽  
Author(s):  
Anne Clancy ◽  
Claire Heride ◽  
Adán Pinto-Fernández ◽  
Andreas Kallinos ◽  
Katherine J. Kayser-Bricker ◽  
...  
Keyword(s):  
Protein Stability ◽  
E3 Ligases ◽  
Molecule Inhibitor ◽  
Steady State Levels ◽  
Chemical Inhibition ◽  
The Stability ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Inhibitor Treatment ◽  
Hct116 Cells

AbstractWhen a ribosome stalls during translation, it runs the risk of collision with a trailing ribosome. Such an encounter leads to the formation of a stable di-ribosome complex, which needs to be resolved by a dedicated machinery. The initial stalling and the subsequent resolution of di-ribosomal complexes requires activity of Makorin and ZNF598 ubiquitin E3 ligases respectively, through ubiquitylation of the eS10 and uS10 sub-units of the ribosome. It is common for the stability of RING E3 ligases to be regulated by an interacting deubiquitylase (DUB), which often opposes auto-ubiquitylation of the E3. Here, we show that the DUB USP9X directly interacts with ZNF598 and regulates its abundance through the control of protein stability in human cells. We have developed a highly specific small molecule inhibitor of USP9X. Proteomics analysis, following inhibitor treatment of HCT116 cells, confirms previous reports linking USP9X with centrosome associated protein stability and reveals loss of ZNF598 and Makorin 2. In the absence of USP9X or following chemical inhibition of its catalytic activity, steady state levels of Makorins and ZNF598 are diminished and the ribosomal quality control pathway is impaired.

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

2018 ◽  
Vol 475 (9) ◽  
pp. 1569-1582 ◽  
Author(s):  
Adaitya Prasad Behera ◽  
Pritam Naskar ◽  
Shubhangi Agarwal ◽  
Prerana Agarwal Banka ◽  
Asim Poddar ◽  
...  
Keyword(s):  
Dissociation Constants ◽  
E3 Ligase ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
New Gene ◽  
Functional Analyses ◽  
Affinity Interaction ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Structural Insights

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.

scholarly journals Arabidopsis DREB2A-Interacting Proteins Function as RING E3 Ligases and Negatively Regulate Plant Drought Stress–Responsive Gene Expression

2008 ◽  
Vol 20 (6) ◽  
pp. 1693-1707 ◽  
Author(s):  
Feng Qin ◽  
Yoh Sakuma ◽  
Lam-Son Phan Tran ◽  
Kyonoshin Maruyama ◽  
Satoshi Kidokoro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drought Stress ◽  
Interacting Proteins ◽  
E3 Ligases ◽  
Responsive Gene ◽  
Ring E3 Ligases ◽  
Ring E3

scholarly journals A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽  
2019 ◽  
Vol 365 (6448) ◽  
pp. eaaw4912 ◽  
Author(s):  
Richard T. Timms ◽  
Zhiqian Zhang ◽  
David Y. Rhee ◽  
J. Wade Harper ◽  
Itay Koren ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Stability ◽  
Protein Degradation ◽  
E3 Ligase ◽  
Cleavage Sites ◽  
Caspase Cleavage ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
The Stability ◽  
Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

scholarly journals Identification of a PGXPP degron motif in dishevelled and structural basis for its binding to the E3 ligase KLHL12

Open Biology ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 200041 ◽  
Author(s):  
Zhuoyao Chen ◽  
Gregory A. Wasney ◽  
Sarah Picaud ◽  
Panagis Filippakopoulos ◽  
Masoud Vedadi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Hydrophobic Interactions ◽  
E3 Ubiquitin Ligase ◽  
Structural Basis ◽  
E3 Ligases ◽  
Kelch Domain ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
New Protein

Wnt signalling is dependent on dishevelled proteins (DVL1-3), which assemble an intracellular Wnt signalosome at the plasma membrane. The levels of DVL1-3 are regulated by multiple Cullin-RING E3 ligases that mediate their ubiquitination and degradation. The BTB-Kelch protein KLHL12 was the first E3 ubiquitin ligase to be identified for DVL1-3, but the molecular mechanisms determining its substrate interactions have remained unknown. Here, we mapped the interaction of DVL1-3 to a ‘PGXPP' motif that is conserved in other known partners and substrates of KLHL12, including PLEKHA4, PEF1, SEC31 and DRD4. To determine the binding mechanism, we solved a 2.4 Å crystal structure of the Kelch domain of KLHL12 in complex with a DVL1 peptide that bound with low micromolar affinity. The DVL1 substrate adopted a U-shaped turn conformation that enabled hydrophobic interactions with all six blades of the Kelch domain β-propeller. In cells, the mutation or deletion of this motif reduced the binding and ubiquitination of DVL1 and increased its stability confirming this sequence as a degron motif for KLHL12 recruitment. These results define the molecular mechanisms determining DVL regulation by KLHL12 and establish the KLHL12 Kelch domain as a new protein interaction module for a novel proline-rich motif.

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

2019 ◽  
Vol 476 (10) ◽  
pp. 1465-1482 ◽  
Author(s):  
Sayani Sarkar ◽  
Adaitya Prasad Behera ◽  
Prateeka Borar ◽  
Prerana Agarwal Banka ◽  
Ajit B. Datta
Keyword(s):  
Enzymatic Activity ◽  
Tryptophan Residue ◽  
Amino Acid Residues ◽  
Binding Studies ◽  
E3 Ligases ◽  
Ring Domains ◽  
Biochemical Analyses ◽  
Ring E3 Ligases ◽  
Ring E3 ◽  
Existing Data

Abstract 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.

scholarly journals Allosteric network in Ube2T drives specificity for RING E3 catalysed ubiquitin signals

2018 ◽  
Author(s):  
Viduth K Chaugule ◽  
Connor Arkinson ◽  
Rachel Toth ◽  
Helen Walden
Keyword(s):  
Dna Damage ◽  
Repair Pathway ◽  
Core Complex ◽  
Site Specific ◽  
E3 Ligases ◽  
Interstrand Crosslink ◽  
Damage Repair ◽  
Molecular Signals ◽  
Ring E3 Ligases ◽  
Ring E3

AbstractIn eukaryotes, DNA damage repair is implemented by a host of proteins that are coordinated by defined molecular signals. One such signal that transpires during the Fanconi Anemia (FA) - interstrand crosslink (ICL) repair pathway is the site-specific monoubiquitination of FANCD2 and FANCI proteins by a large, multi-protein FA core complex. The mechanics for this exquisitely specific monoubiquitin signal has been elusive. Here we show FANCL, the RING E3 module of the FA core complex, allosterically activates its cognate E2 Ube2T for monoubiquitination by a mechanism distinct from the typical RING-based catalysis. FANCL triggers intricate re-wiring of Ube2T’s intra-residue network thus activating the E2 for precision targeting. This network is intrinsically regulated by conserved gates and loops which can be engineered to yield Ube2T variants that enhance FANCD2 ubiquitination by ~30-fold without compromising on target specificity. Finally, we also uncover allosteric networks in other ubiquitin E2s that can be leveraged by RING E3 ligases to drive specific ubiquitination.

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