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1.  RBR E3 ubiquitin ligases: new structures, new insights, new questions 
Biochemical Journal  2014;458(Pt 3):421-437.
The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinson's disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.
doi:10.1042/BJ20140006
PMCID: PMC3940038  PMID: 24576094
catalysis; structure; ubiquitination; ubiquitin ligase; ANKIB1, ankyrin repeat- and IBR domain-containing 1; BRcat, benign-catalytic; CCCP, carbonyl cyanide m-chlorophenylhydrazone; Cdk5, cyclin-dependent kinase 5; cIAP2, cellular inhibitor of apoptosis 2; CK1, casein kinase 1; CPH, Cul7, Parc and HERC2 proteins; CRL, Cul-RING-ligase; Cul, cullin; Eps15, epidermal growth factor receptor pathway substrate 15; FANCL, Fanconi anaemia, complementation group L; HDAC, histone deacetylase; HECT, homologous with E6-associated protein C-terminus; HOIL-1, haem-oxidized IRP2 ubiquitin ligase 1; HOIP, HOIL-1-interacting protein; IBR, InBetweenRING; LUBAC, linear ubiquitin chain assembly complex; MDM2, murine double minute 2; MIRO, mitochondrial Rho GTPase; NEDD, neural-precursor-cell-expressed developmentally down-regulated; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; NZF, Npl4 ZNF; Parc, parkin-like cytoplasmic p53-binding protein; PINK1, PTEN-induced putative kinase 1; PKC, protein kinase C; RanBP2, RAN-binding protein 2; RBR, RING-BetweenRING-RING/RING1-BRcat-Rcat; Rcat, required-for-catalysis; RNF, RING finger protein; RWD, RING finger and WD repeat-containing; SH3, Src homology 3; SHARPIN, SHANK-associated RH domain interactor; SILAC, stable isotope labelling by amino acids in cell culture; SUMO, small ubiquitin-related modifier; TOMM70A, translocase of outer mitochondrial membrane 70 homologue A; TRAF6, tumour-necrosis-factor-receptor-associated factor 6; TRIAD, two RING fingers and a DRIL (double RING finger linked); UBA, ubiquitin-associated; UBE2L, ubiquitin-conjugating enzyme E2L; UIM, ubiquitin-interacting motif; Ubl, ubiquitin-like; ZNF, zinc finger
2.  Structure of the Human FANCL RING-Ube2T Complex Reveals Determinants of Cognate E3-E2 Selection 
Structure(London, England:1993)  2014;22(2):337-344.
Summary
The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL.
Graphical Abstract
Highlights
•Structure of the ubiquitination platform FANCL-Ube2T is essential for DNA ICL repair•Structural basis for cognate E2-E3 enzyme pairing•Interactions beyond the generic E2-E3 interface are required for E2-E3 selectivity
Ube2T and FANCL represent a specific E2-E3 ubiquitin-conjugating enzyme and ubiquitin ligase pair required for the function of the Fanconi anemia DNA repair pathway. Hodson et al. report a structure of Ube2T in complex with the RING domain of FANCL and reveal molecular determinants of E2-E3 specificity.
doi:10.1016/j.str.2013.12.004
PMCID: PMC3979106  PMID: 24389026
3.  Molecular replacements 
An introduction to the proceedings of the CCP4 Study Weekend held at the East Midlands Conference Centre of the University of Nottingham, England, in January 2013.
doi:10.1107/S0907444913027352
PMCID: PMC3817688  PMID: 24189226
CCP4 Study Weekend 2013
4.  Towards a Molecular Understanding of the Fanconi Anemia Core Complex 
Anemia  2012;2012:926787.
Fanconi Anemia (FA) is a genetic disorder characterized by the inability of patient cells to repair DNA damage caused by interstrand crosslinking agents. There are currently 14 verified FA genes, where mutation of any single gene prevents repair of DNA interstrand crosslinks (ICLs). The accumulation of ICL damage results in genome instability and patients having a high predisposition to cancers. The key event of the FA pathway is dependent on an eight-protein core complex (CC), required for the monoubiquitination of each member of the FANCD2-FANCI complex. Interestingly, the majority of patient mutations reside in the CC. The molecular mechanisms underlying the requirement for such a large complex to carry out a monoubiquitination event remain a mystery. This paper documents the extensive efforts of researchers so far to understand the molecular roles of the CC proteins with regard to its main function in the FA pathway, the monoubiquitination of FANCD2 and FANCI.
doi:10.1155/2012/926787
PMCID: PMC3364535  PMID: 22675617
5.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 
Open Biology  2012;2(5):120080.
Summary
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
doi:10.1098/rsob.120080
PMCID: PMC3376738  PMID: 22724072
PINK1; Parkin; Parkinson's disease
6.  Small, N-Terminal Tags Activate Parkin E3 Ubiquitin Ligase Activity by Disrupting Its Autoinhibited Conformation 
PLoS ONE  2012;7(4):e34748.
Parkin is an E3 ubiquitin ligase, mutations in which cause Autosomal Recessive Parkinson's Disease. Many studies aimed at understanding Parkin function, regulation and dysfunction are performed using N-terminal epitope tags. We report here that the use of small tags such as FLAG, cMyc and HA, influence the physical stability and activity of Parkin in and out of cells, perturbing the autoinhibited native state of Parkin, resulting in an active-for-autoubiquitination species.
doi:10.1371/journal.pone.0034748
PMCID: PMC3319606  PMID: 22496854
7.  Structural Analysis of Human FANCL, the E3 Ligase in the Fanconi Anemia Pathway* 
The Journal of Biological Chemistry  2011;286(37):32628-32637.
The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein “core complex” containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.
doi:10.1074/jbc.M111.244632
PMCID: PMC3173227  PMID: 21775430
Crystal Structure; Crystallography; DNA Damage; E3 Ubiquitin Ligase; Protein Structure
8.  The structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex 
The Fanconi Anemia pathway is activated in response to DNA damage, leading to monoubiquitination of the substrates FANCI and FANCD2 by the Fanconi Anemia core complex. Here we report the crystal structure of FANCL, the catalytic subunit of the Fanconi Anemia core complex at 3.2 Å. The structure reveals an architecture that is fundamentally different from previous sequence-based predictions. The molecule is composed of an N-terminal E2-like fold, which we term the ELF domain, a novel double-RWD (DRWD) domain, and a C-terminal RING domain predicted to facilitate E2 binding. Binding assays demonstrate that the DRWD domain, but not the ELF domain, is responsible for substrate binding.
doi:10.1038/nsmb.1759
PMCID: PMC2929457  PMID: 20154706
9.  Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases 
Molecular cell  2009;36(1):39-50.
SUMMARY
In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC), and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call “3-box” due to its facilitating Cul3-binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3, and how their dimerization and conformational variability may facilitate avid interactions with diverse substrates.
doi:10.1016/j.molcel.2009.09.022
PMCID: PMC2847577  PMID: 19818708
10.  Selenium incorporation using recombinant techniques 
An overview of techniques for recombinant incorporation of selenium and subsequent purification and crystallization of the resulting labelled protein.
Using selenomethionine to phase macromolecular structures is common practice in structure determination, along with the use of selenocysteine. Selenium is consequently the most commonly used heavy atom for MAD. In addition to the well established recombinant techniques for the incorporation of selenium in pro­karyal expression systems, there have been recent advances in selenium labelling in eukaryal expression, which will be discussed. Tips and things to consider for the purification and crystallization of seleno-labelled proteins are also included.
doi:10.1107/S0907444909038207
PMCID: PMC2852298  PMID: 20382987
selenium incorporation; selenium labelling
11.  A molecular explanation for the recessive nature of parkin-linked Parkinson’s disease 
Nature Communications  2013;4:1983.
Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.
Mutations in the E3 ubiquitin ligase parkin are associated with juvenile Parkinson’s disease. Here the authors report the solution structure of the Parkin RING2 domain, revealing how disease-associated mutations affect its function and providing a molecular explanation for the recessive nature of the disease.
doi:10.1038/ncomms2983
PMCID: PMC3709501  PMID: 23770917

Results 1-11 (11)