scholarly journals Structural Basis of Ubiquitin Recognition by the Ubiquitin-associated (UBA) Domain of the Ubiquitin Ligase EDD

2007 ◽  
Vol 282 (49) ◽  
pp. 35787-35795 ◽  
Author(s):  
Guennadi Kozlov ◽  
Long Nguyen ◽  
Tong Lin ◽  
Gregory De Crescenzo ◽  
Morag Park ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Damage Repair ◽  
The Family ◽  
Uba Domain ◽  
Ordered Water

EDD (or HYD) is an E3 ubiquitin ligase in the family of HECT (homologous to E6-AP C terminus) ligases. EDD contains an N-terminal ubiquitin-associated (UBA) domain, which is present in a variety of proteins involved in ubiquitin-mediated processes. Here, we use isothermal titration calorimetry (ITC), NMR titrations, and pull-down assays to show that the EDD UBA domain binds ubiquitin. The 1.85Å crystal structure of the complex with ubiquitin reveals the structural basis of ubiquitin recognition by UBA helices α1 and α3. The structure shows a larger number of intermolecular hydrogen bonds than observed in previous UBA/ubiquitin complexes. Two of these involve ordered water molecules. The functional importance of residues at the UBA/ubiquitin interface was confirmed using site-directed mutagenesis. Surface plasmon resonance (SPR) measurements show that the EDD UBA domain does not have a strong preference for polyubiquitin chains over monoubiquitin. This suggests that EDD binds to monoubiquitinated proteins, which is consistent with its involvement in DNA damage repair pathways.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP21

2008 ◽  
Vol 417 (1) ◽  
pp. 149-165 ◽  
Author(s):  
John W. Blankenship ◽  
Eugene Varfolomeev ◽  
Tatiana Goncharov ◽  
Anna V. Fedorova ◽  
Donald S. Kirkpatrick ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Proteasomal Degradation ◽  
Nuclear Factor Κb ◽  
Titration Calorimetry ◽  
Tumour Necrosis Factor Receptor ◽  
Amino Acid Residues ◽  
Polyubiquitin Chains ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Binding ◽  
Uba Domain

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-κB (nuclear factor κB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-κB-inducing kinase) and regulate the non-canonical NF-κB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys48- and Lys63-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain–ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile44 of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

scholarly journals The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells

2019 ◽  
Vol 116 (27) ◽  
pp. 13293-13298 ◽  
Author(s):  
Ian R. Kelsall ◽  
Jiazhen Zhang ◽  
Axel Knebel ◽  
J. Simon C. Arthur ◽  
Philip Cohen
Keyword(s):  
Ubiquitin Ligase ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
De Novo ◽  
E3 Ligase ◽  
Toll Like Receptor ◽  
Signaling Mechanism ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
Physiological Substrates

The linear ubiquitin assembly complex (LUBAC) comprises 3 components: HOIP, HOIL-1, and Sharpin, of which HOIP and HOIL-1 are both members of the RBR subfamily of E3 ubiquitin ligases. HOIP catalyses the formation of Met1-linked ubiquitin oligomers (also called linear ubiquitin), but the function of the E3 ligase activity of HOIL-1 is unknown. Here, we report that HOIL-1 is an atypical E3 ligase that forms oxyester bonds between the C terminus of ubiquitin and serine and threonine residues in its substrates. Exploiting the sensitivity of HOIL-1–generated oxyester bonds to cleavage by hydroxylamine, and macrophages from knock-in mice expressing the E3 ligase-inactive HOIL-1[C458S] mutant, we identify IRAK1, IRAK2, and MyD88 as physiological substrates of the HOIL-1 E3 ligase during Toll-like receptor signaling. HOIL-1 is a monoubiquitylating E3 ubiquitin ligase that initiates the de novo synthesis of polyubiquitin chains that are attached to these proteins in macrophages. HOIL-1 also catalyses its own monoubiquitylation in cells and most probably the monoubiquitylation of Sharpin, in which ubiquitin is also attached by an oxyester bond. Our study establishes that oxyester-linked ubiquitylation is used as an intracellular signaling mechanism.

scholarly journals Family D DNA polymerase interacts with GINS to promote CMG-helicase in the archaeal replisome

2021 ◽  
Author(s):  
Keisuke Oki ◽  
Mariko Nagata ◽  
Takeshi Yamagami ◽  
Tomoyuki Numata ◽  
Sonoko Ishino ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Dna Polymerase ◽  
Direct Interaction ◽  
Crystal Structure Analysis ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Functional Connection ◽  
Terminal Domain ◽  
The Family ◽  
Lagging Strand

Abstract Genomic DNA replication requires replisome assembly. We show here the molecular mechanism by which CMG (GAN–MCM–GINS)-like helicase cooperates with the family D DNA polymerase (PolD) in Thermococcus kodakarensis. The archaeal GINS contains two Gins51 subunits, the C-terminal domain of which (Gins51C) interacts with GAN. We discovered that Gins51C also interacts with the N-terminal domain of PolD’s DP1 subunit (DP1N) to connect two PolDs in GINS. The two replicases in the replisome should be responsible for leading- and lagging-strand synthesis, respectively. Crystal structure analysis of the DP1N–Gins51C–GAN ternary complex was provided to understand the structural basis of the connection between the helicase and DNA polymerase. Site-directed mutagenesis analysis supported the interaction mode obtained from the crystal structure. Furthermore, the assembly of helicase and replicase identified in this study is also conserved in Eukarya. PolD enhances the parental strand unwinding via stimulation of ATPase activity of the CMG-complex. This is the first evidence of the functional connection between replicase and helicase in Archaea. These results suggest that the direct interaction of PolD with CMG-helicase is critical for synchronizing strand unwinding and nascent strand synthesis and possibly provide a functional machinery for the effective progression of the replication fork.

scholarly journals Zinc Binding Properties of Engineered RING Finger Domain of Arkadia E3 Ubiquitin Ligase

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Christos T. Chasapis ◽  
Ariadni K. Loutsidou ◽  
Malvina G. Orkoula ◽  
Georgios A. Spyroulias
Keyword(s):  
Ubiquitin Ligase ◽  
Structural Integrity ◽  
Recombinant Expression ◽  
E3 Ubiquitin Ligase ◽  
Ring Finger ◽  
Ring Domain ◽  
Site Directed Mutagenesis ◽  
Zinc Binding ◽  
Ring Finger Domain ◽  
C Terminus

Human Arkadia is a nuclear protein consisted of 989 amino acid residues, with a characteristic RING domain in its C-terminus. The RING domain harbours the E3 ubiquitin ligase activity needed by Arkadia to ubiquitinate its substrates such as negative regulators of TGF- signaling. The RING finger domain of Arkadia is a RING-H2 type and its structure and stability is strongly dependent on the presence of two bound Zn(II) ions attached to the protein frame through a defined Cys3-His2-Cys3 motif. In the present paper we transform the RING-H2 type of Arkadia finger domain to nonnative RING sequence, substituting the zinc-binding residues or to Arginine, through site-directed mutagenesis. The recombinant expression, inEscherichia coli, of the mutants C955R and H960R reveal significant lower yield in respect with the native polypeptide of Arkadia RING-H2 finger domain. In particular, only the C955R mutant exhibits expression yield sufficient for recombinant protein isolation and preliminary studies. Atomic absorption measurements and preliminary NMR data analysis reveal that the C955R point mutation in the RING Finger domain of Arkadia diminishes dramatically the zinc binding affinity, leading to the breakdown of the global structural integrity of the RING construct.

Generation of free ubiquitin chains is up-regulated in stress and facilitated by the HECT domain ubiquitin ligases UFD4 and HUL5

2012 ◽  
Vol 444 (3) ◽  
pp. 611-617 ◽  
Author(s):  
Ori Braten ◽  
Nitzan Shabek ◽  
Yelena Kravtsova-Ivantsiv ◽  
Aaron Ciechanover
Keyword(s):  
Signal Transduction ◽  
Dna Damage ◽  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Specific Substrate ◽  
Polyubiquitin Chains ◽  
Hect Domain ◽  
Protein Substrate ◽  
C Terminus ◽  
Free Ubiquitin

Polyubiquitin chains serve a variety of physiological roles. Typically the chains are bound covalently to a protein substrate and in many cases target it for degradation by the 26S proteasome. However, several studies have demonstrated the existence of free polyubiquitin chains which are not linked to a specific substrate. Several physiological functions have been attributed to these chains, among them playing a role in signal transduction and serving as storage of ubiquitin for utilization under stress. In the present study, we have established a system for the detection of free ubiquitin chains and monitoring their level under changing conditions. Using this system, we show that UFD4 (ubiquitin fusion degradation 4), a HECT (homologous with E6-AP C-terminus) domain ubiquitin ligase, is involved in free chain generation. We also show that generation of these chains is stimulated in response to a variety of stresses, particularly those caused by DNA damage. However, it appears that the stress-induced synthesis of free chains is catalysed by a different ligase, HUL5 (HECT ubiquitin ligase 5), which is also a HECT domain E3.

scholarly journals The Structural Basis of the Binding of Various Aminopolycarboxylates by the Periplasmic EDTA-Binding Protein EppA from Chelativorans sp. BNC1

2020 ◽  
Vol 21 (11) ◽  
pp. 3940 ◽  
Author(s):  
Kevin M. Lewis ◽  
Chelsie L. Greene ◽  
Steven A. Sattler ◽  
Buhyun Youn ◽  
Luying Xun ◽  
...  
Keyword(s):  
Binding Protein ◽  
Metal Chelate ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Bacterial Cells ◽  
Chelate Complexes ◽  
Quantum Chemical Analysis ◽  
Ligand Selectivity

The widespread use of synthetic aminopolycarboxylates, such as ethylenediaminetetraacetate (EDTA), as chelating agents has led to their contamination in the environment as stable metal–chelate complexes. Microorganisms can transport free EDTA, but not metal–EDTA complexes, into cells for metabolism. An ABC-type transporter for free EDTA uptake in Chelativorans sp. BNC1 was investigated to understand the mechanism of the ligand selectivity. We solved the X-ray crystal structure of the periplasmic EDTA-binding protein (EppA) and analyzed its structure–function relations through isothermal titration calorimetry, site-directed mutagenesis, molecular docking, and quantum chemical analysis. EppA had high affinities for EDTA and other aminopolycarboxylates, which agrees with structural analysis, showing that its binding pocket could accommodate free aminopolycarboxylates. Further, key amino acid residues involved in the binding were identified. Our results suggest that EppA is a general binding protein for the uptake of free aminopolycarboxylates. This finding suggests that bacterial cells import free aminopolycarboxylates, explaining why stable metal–chelate complexes are resistant to degradation, as they are not transported into the cells for degradation.

scholarly journals Insights into the Function of theN-Acetyltransferase SatA That Detoxifies Streptothricin inBacillus subtilisandBacillus anthracis

2019 ◽  
Vol 85 (6) ◽  
Author(s):  
Rachel M. Burckhardt ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Binding Site ◽  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Side Chains ◽  
Missense Mutations ◽  
Content Type ◽  
C Terminus ◽  
Exclusion Chromatography ◽  
Domains Of Life

ABSTRACTAcylation of epsilon amino groups of lysyl side chains is a widespread modification of proteins and small molecules in cells of all three domains of life. Recently, we showed thatBacillus subtilisandBacillus anthracisencode the GCN5-relatedN-acetyltransferase (GNAT) SatA that can acetylate and inactivate streptothricin, which is a broad-spectrum antibiotic produced by actinomycetes in the soil. To determine functionally relevant residues ofB. subtilisSatA (BsSatA), a mutational screen was performed, highlighting the importance of a conserved area near the C terminus. Upon inspection of the crystal structure of theB. anthracisAmes SatA (BaSatA; PDB entry 3PP9), this area appears to form a pocket with multiple conserved aromatic residues; we hypothesized this region contains the streptothricin-binding site. Chemical and site-directed mutagenesis was used to introduce missense mutations intosatA, and the functionality of the variants was assessed using a heterologous host (Salmonella enterica). Results of isothermal titration calorimetry experiments showed that residue Y164 ofBaSatA was important for binding streptothricin. Results of size exclusion chromatography analyses showed that residue D160 was important for dimerization. Together, these data advance our understanding of how SatA interacts with streptothricin.IMPORTANCEThis work provides insights into how an abundant antibiotic found in soil is bound to the enzyme that inactivates it. This work identifies residues for the binding of the antibiotic and probes the contributions of substituting side chains for those in the native protein, providing information regarding hydrophobicity, size, and flexibility of the antibiotic binding site.

scholarly journals Phytophthora sojae effector Avr1d functions as an E2 competitor and inhibits ubiquitination activity of GmPUB13 to facilitate infection

2021 ◽  
Vol 118 (10) ◽  
pp. e2018312118
Author(s):  
Yachun Lin ◽  
Qinli Hu ◽  
Jia Zhou ◽  
Weixiao Yin ◽  
Deqiang Yao ◽  
...  
Keyword(s):  
Hairy Roots ◽  
Ubiquitin Ligase ◽  
E3 Ligase ◽  
Phytophthora Sojae ◽  
Host Cells ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Susceptibility Factor

Oomycete pathogens such as Phytophthora secrete a repertoire of effectors into host cells to manipulate host immunity and benefit infection. In this study, we found that an RxLR effector, Avr1d, promoted Phytophthora sojae infection in soybean hairy roots. Using a yeast two-hybrid screen, we identified the soybean E3 ubiquitin ligase GmPUB13 as a host target for Avr1d. By coimmunoprecipitation (Co-IP), gel infiltration, and isothermal titration calorimetry (ITC) assays, we confirmed that Avr1d interacts with GmPUB13 both in vivo and in vitro. Furthermore, we found that Avr1d inhibits the E3 ligase activity of GmPUB13. The crystal structure Avr1d in complex with GmPUB13 was solved and revealed that Avr1d occupies the binding site for E2 ubiquitin conjugating enzyme on GmPUB13. In line with this, Avr1d competed with E2 ubiquitin conjugating enzymes for GmPUB13 binding in vitro, thereby decreasing the E3 ligase activity of GmPUB13. Meanwhile, we found that inactivation of the ubiquitin ligase activity of GmPUB13 stabilized GmPUB13 by blocking GmPUB13 degradation. Silencing of GmPUB13 in soybean hairy roots decreased P. sojae infection, suggesting that GmPUB13 acts as a susceptibility factor. Altogether, this study highlights a virulence mechanism of Phytophthora effectors, by which Avr1d competes with E2 for GmPUB13 binding to repress the GmPUB13 E3 ligase activity and thereby stabilizing the susceptibility factor GmPUB13 to facilitate Phytophthora infection. This study unravels the structural basis for modulation of host targets by Phytophthora effectors and will be instrumental for boosting plant resistance breeding.

scholarly journals Structural basis for the binding of tryptophan-based motifs by δ-COP

2015 ◽  
Vol 112 (46) ◽  
pp. 14242-14247 ◽  
Author(s):  
Richard J. Suckling ◽  
Pak Phi Poon ◽  
Sophie M. Travis ◽  
Irina V. Majoul ◽  
Frederick M. Hughson ◽  
...  
Keyword(s):  
Adaptor Protein ◽  
Structural Basis ◽  
Titration Calorimetry ◽  
Carboxypeptidase Y ◽  
Binding Partner ◽  
C Terminus ◽  
Tryptophan Residues ◽  
A Site ◽  
Adp Ribosylation Factor ◽  
Unstructured Regions

Coatomer consists of two subcomplexes: the membrane-targeting, ADP ribosylation factor 1 (Arf1):GTP-binding βγδζ-COP F-subcomplex, which is related to the adaptor protein (AP) clathrin adaptors, and the cargo-binding αβ’ε-COP B-subcomplex. We present the structure of the C-terminal μ-homology domain of the yeast δ-COP subunit in complex with the WxW motif from its binding partner, the endoplasmic reticulum-localized Dsl1 tether. The motif binds at a site distinct from that used by the homologous AP μ subunits to bind YxxΦ cargo motifs with its two tryptophan residues sitting in compatible pockets. We also show that the Saccharomyces cerevisiae Arf GTPase-activating protein (GAP) homolog Gcs1p uses a related WxxF motif at its extreme C terminus to bind to δ-COP at the same site in the same way. Mutations designed on the basis of the structure in conjunction with isothermal titration calorimetry confirm the mode of binding and show that mammalian δ-COP binds related tryptophan-based motifs such as that from ArfGAP1 in a similar manner. We conclude that δ-COP subunits bind Wxn(1–6)[WF] motifs within unstructured regions of proteins that influence the lifecycle of COPI-coated vesicles; this conclusion is supported by the observation that, in the context of a sensitizing domain deletion in Dsl1p, mutating the tryptophan-based motif-binding site in yeast causes defects in both growth and carboxypeptidase Y trafficking/processing.

scholarly journals The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation

2000 ◽  
Vol 20 (4) ◽  
pp. 1382-1393 ◽  
Author(s):  
Kenneth Wu ◽  
Serge Y. Fuchs ◽  
Angus Chen ◽  
Peilin Tan ◽  
Carlos Gomez ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Binding Activity ◽  
Dual Function ◽  
Necrosis Factor Alpha ◽  
Polyubiquitin Chains ◽  
C Terminus ◽  
N Terminus ◽  
Factor Alpha ◽  
F Box Protein ◽  
Necessary And Sufficient

ABSTRACT We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

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