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

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.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.735681965.793570379 ◽

2020 ◽

Author(s):

Thorsten Hoppe

Keyword(s):

E3 Ligases ◽

Ring E3 Ligases ◽

Ring E3

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

The Plant Cell ◽

10.1105/tpc.107.057380 ◽

2008 ◽

Vol 20 (6) ◽

pp. 1693-1707 ◽

Cited By ~ 285

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

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

Open Biology ◽

10.1098/rsob.200041 ◽

2020 ◽

Vol 10 (6) ◽

pp. 200041 ◽

Cited By ~ 1

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.

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

10.1101/429076 ◽

2018 ◽

Cited By ~ 2

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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Flexible Cullins in Cullin-RING E3 Ligases Allosterically Regulate Ubiquitination

Journal of Biological Chemistry ◽

10.1074/jbc.m111.277236 ◽

2011 ◽

Vol 286 (47) ◽

pp. 40934-40942 ◽

Cited By ~ 37

Author(s):

Jin Liu ◽

Ruth Nussinov

Keyword(s):

E3 Ligases ◽

Ring E3 Ligases ◽

Ring E3

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Keratin 6a reorganization for ubiquitin–proteasomal processing is a direct antimicrobial response

The Journal of Cell Biology ◽

10.1083/jcb.201704186 ◽

2017 ◽

Vol 217 (2) ◽

pp. 731-744 ◽

Cited By ~ 9

Author(s):

Jonathan K.L. Chan ◽

Don Yuen ◽

Priscilla Hiu-Mei Too ◽

Yan Sun ◽

Belinda Willard ◽

...

Keyword(s):

Ubiquitin Proteasome System ◽

Immune Defense ◽

Antigenic Peptides ◽

Corneal Epithelial ◽

E3 Ligases ◽

Appreciable Increase ◽

Host Defense Response ◽

Ubiquitin Proteasome ◽

Ring E3 Ligases ◽

Ring E3

Skin and mucosal epithelia deploy antimicrobial peptides (AMPs) to eliminate harmful microbes. We reported that the intermediate filament keratin 6a (K6a) is constitutively processed into antimicrobial fragments in corneal epithelial cells. In this study, we show that K6a network remodeling is a host defense response that directly up-regulates production of keratin-derived AMPs (KAMPs) by the ubiquitin–proteasome system (UPS). Bacterial ligands trigger K6a phosphorylation at S19, S22, S37, and S60, leading to network disassembly. Mutagenic analysis of K6a confirmed that the site-specific phosphorylation augmented its solubility. K6a in the cytosol is ubiquitinated by cullin-RING E3 ligases for subsequent proteasomal processing. Without an appreciable increase in K6a gene expression and proteasome activity, a higher level of cytosolic K6a results in enhanced KAMP production. Although proteasome-mediated proteolysis is known to produce antigenic peptides in adaptive immunity, our findings demonstrate its new role in producing AMPs for innate immune defense. Manipulating K6a phosphorylation or UPS activity may provide opportunities to harness the innate immunity of epithelia against infection.

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The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases

Molecular & Cellular Proteomics ◽

10.1074/mcp.ra120.002414 ◽

2020 ◽

pp. mcp.RA120.002414

Author(s):

Ryan J Lumpkin ◽

Alla S Ahmad ◽

Rachel Blake ◽

Christopher J Condon ◽

Elizabeth A. Komives

Keyword(s):

Mass Spectrometry ◽

Binding Site ◽

Mass Spectrometry Analysis ◽

E3 Ligases ◽

Spectrometry Analysis ◽

Exchange Mass Spectrometry ◽

Hydrogen Deuterium ◽

Ring E3 Ligases ◽

Ring E3

Cullin RING E3 Ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 Ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs) and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contribute to polyubiquitylation but do not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

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