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1.
Eur Biophys J ; 2021 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-33609147

RESUMO

Lysins are a class of hydrolytic enzymes used by bacteriophages to target and cleave the peptidoglycan of bacterial cell walls during their lytic cycle. The lysins from bacteriophages that infect Gram-positive bacteria are typically monomeric and consist of one or two catalytic domains (CD) and a cell binding domain (CBD). However, multimeric lysins encoded by a single gene have also been reported, among which Lys170 from enterococcal phage F170/08 was one of the first identified. Here, we determined the crystal structure of Lys170 CBD at 1.40 Å resolution. The structure reveals that Lys170 CBDs assemble into a tetrameric functional unit and that each monomer folds into a three-stranded ß-sheet core capped on each side by an α-helix. In addition, we identified key residues of Lys170 CBD involved in host cell binding. Our work provides a basis for designing highly efficient lysins targeting Enterococcus faecalis.

2.
Elife ; 92020 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-33136002

RESUMO

Legionella pneumophila extensively modulates the host ubiquitin network to create the Legionella-containing vacuole (LCV) for its replication. Many of its virulence factors function as ubiquitin ligases or deubiquitinases (DUBs). Here, we identify Lem27 as a DUB that displays a preference for diubiquitin formed by K6, K11, or K48. Lem27 is associated with the LCV where it regulates Rab10 ubiquitination in concert with SidC and SdcA, two bacterial E3 ubiquitin ligases. Structural analysis of the complex formed by an active fragment of Lem27 and the substrate-based suicide inhibitor ubiquitin-propargylamide (PA) reveals that it harbors a fold resembling those in the OTU1 DUB subfamily with a Cys-His catalytic dyad and that it recognizes ubiquitin via extensive hydrogen bonding at six contact sites. Our results establish Lem27 as a DUB that functions to regulate protein ubiquitination on L. pneumophila phagosomes by counteracting the activity of bacterial ubiquitin E3 ligases.

3.
Nucleic Acids Res ; 48(19): 11054-11067, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33045733

RESUMO

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.


Assuntos
Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Shewanella/metabolismo , Sistemas Toxina-Antitoxina
4.
PLoS Pathog ; 16(3): e1008394, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32176738

RESUMO

Using bacteriophage-derived endolysins as an alternative strategy for fighting drug-resistant bacteria has recently been garnering renewed interest. However, their application is still hindered by their narrow spectra of activity. In our previous work, we demonstrated that the endolysin LysIME-EF1 possesses efficient bactericidal activity against multiple strains of Enterococcus faecalis (E. faecalis). Herein, we observed an 8 kDa fragment and hypothesized that it contributes to LysIME-EF1 lytic activity. To examine our hypothesis, we determined the structure of LysIME-EF1 at 1.75 Å resolution. LysIME-EF1 exhibits a unique architecture in which one full-length LysIME-EF1 forms a tetramer with three additional C-terminal cell-wall binding domains (CBDs) that correspond to the abovementioned 8 kDa fragment. Furthermore, we identified an internal ribosomal binding site (RBS) and alternative start codon within LysIME-EF1 gene, which are demonstrated to be responsible for the translation of the truncated CBD. To elucidate the molecular mechanism for the lytic activity of LysIME-EF1, we combined mutagenesis, lytic activity assays and in vivo animal infection experiments. The results confirmed that the additional LysIME-EF1 CBDs are important for LysIME-EF1 architecture and its lytic activity. To our knowledge, this is the first determined structure of multimeric endolysin encoded by a single gene in E. faecalis phages. As such, it may provide valuable insights into designing potent endolysins against the opportunistic pathogen E. faecalis.


Assuntos
Bacteriófagos/química , Endopeptidases/química , Enterococcus faecalis/virologia , Genes Virais , Proteínas Virais/química , Bacteriófagos/genética , Cristalografia por Raios X , Endopeptidases/genética , Enterococcus faecalis/química , Domínios Proteicos , Proteínas Virais/genética
5.
J Biol Chem ; 295(6): 1646-1657, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-31907282

RESUMO

Legionella pneumophila is the causative agent of the lung malady Legionnaires' disease, it modulates host function to create a niche termed the Legionella-containing vacuole (LCV) that permits intracellular L. pneumophila replication. One important aspect of such modulation is the co-option of the host ubiquitin network with a panel of effector proteins. Here, using recombinantly expressed and purified proteins, analytic ultracentrifugation, structural analysis, and computational modeling, along with deubiquitinase (DUB), and bacterial infection assays, we found that the bacterial defective in organelle trafficking/intracellular multiplication effector Ceg23 is a member of the ovarian tumor (OTU) DUB family. We found that Ceg23 displays high specificity toward Lys-63-linked polyubiquitin chains and is localized on the LCV, where it removes ubiquitin moieties from proteins ubiquitinated by the Lys-63-chain type. Analysis of the crystal structure of a Ceg23 variant lacking two putative transmembrane domains at 2.80 Å resolution revealed that despite very limited homology to established members of the OTU family at the primary sequence level, Ceg23 harbors a catalytic motif resembling those associated with typical OTU-type DUBs. ceg23 deletion increased the association of Lys-63-linked polyubiquitin with the bacterial phagosome, indicating that Ceg23 regulates Lys-63-linked ubiquitin signaling on the LCV. In summary, our findings indicate that Ceg23 contributes to the regulation of the association of Lys-63 type polyubiquitin with the Legionella phagosome. Future identification of host substrates targeted by Ceg23 could clarify the roles of these polyubiquitin chains in the intracellular life cycle of L. pneumophila and Ceg23's role in bacterial virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas Desubiquitinantes/metabolismo , Legionella pneumophila/metabolismo , Doença dos Legionários/microbiologia , Poliubiquitina/metabolismo , Proteínas de Bactérias/química , Enzimas Desubiquitinantes/química , Células HEK293 , Células HeLa , Humanos , Legionella pneumophila/química , Doença dos Legionários/metabolismo , Lisina/metabolismo , Fagossomos/metabolismo , Conformação Proteica , Especificidade por Substrato , Ubiquitinação
6.
Nature ; 572(7769): 387-391, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31330531

RESUMO

The bacterial pathogen Legionella pneumophila creates an intracellular niche permissive for its replication by extensively modulating host-cell functions using hundreds of effector proteins delivered by its Dot/Icm secretion system1. Among these, members of the SidE family (SidEs) regulate several cellular processes through a unique phosphoribosyl ubiquitination mechanism that bypasses the canonical ubiquitination machinery2-4. The activity of SidEs is regulated by another Dot/Icm effector known as SidJ5; however, the mechanism of this regulation is not completely understood6,7. Here we demonstrate that SidJ inhibits the activity of SidEs by inducing the covalent attachment of glutamate moieties to SdeA-a member of the SidE family-at E860, one of the catalytic residues that is required for the mono-ADP-ribosyltransferase activity involved in ubiquitin activation2. This inhibition by SidJ is spatially restricted in host cells because its activity requires the eukaryote-specific protein calmodulin (CaM). We solved a structure of SidJ-CaM in complex with AMP and found that the ATP used in this reaction is cleaved at the α-phosphate position by SidJ, which-in the absence of glutamate or modifiable SdeA-undergoes self-AMPylation. Our results reveal a mechanism of regulation in bacterial pathogenicity in which a glutamylation reaction that inhibits the activity of virulence factors is activated by host-factor-dependent acyl-adenylation.


Assuntos
Calmodulina/metabolismo , Ácido Glutâmico/metabolismo , Legionella pneumophila/enzimologia , Legionella pneumophila/metabolismo , Ubiquitinação , ADP-Ribosilação , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Coenzimas/metabolismo , Células HEK293 , Humanos , Legionella pneumophila/citologia , Modelos Moleculares , Ubiquitina/química , Ubiquitina/metabolismo
8.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 12): 713, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29199994

RESUMO

An extra affiliation is added for the authors of the article by Wu et al. [(2017), Acta Cryst. F73, 393-397].

9.
Acta Crystallogr F Struct Biol Commun ; 73(Pt 7): 393-397, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28695847

RESUMO

The myelin sheath, which envelops axons in the vertebrate central nervous system, is crucial for the rapid conduction of action potentials. Myelin-gene regulatory factor (MRF) is a recently identified transcription factor that is required for myelin-sheath formation. Loss of MRF leads to demyelinating diseases and motor learning deficiency. MRF is a membrane-bound transcription factor that undergoes autocleavage from the endoplasmic reticulum membrane. The N-terminus of MRF contains a DNA-binding domain (DBD) that functions as a homotrimer. In this study, the MRF DBD was cloned, purified and crystallized in order to understand the molecular mechanism that regulates the transcription of myelin genes. Selenomethionine was subsequently introduced into the crystals to obtain the phases for the MRF DBD structure. The native and selenomethionine-labelled crystals exhibited diffraction to 2.50 and 2.51 Šresolution, respectively. The crystals belonged to space group P321 and the selenomethionine-labelled crystals had unit-cell parameters a = 104.0, b = 104.0, c = 46.7 Å, α = 90, ß = 90, γ = 120°. The calculated Matthews coefficient was 3.04 Å3 Da-1 and the solvent content was 59.5%, indicating the presence of one MRF DBD molecule in the asymmetric unit.


Assuntos
Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Camundongos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Fatores de Transcrição/genética , Fatores de Transcrição/isolamento & purificação , Difração de Raios X
10.
Sci Rep ; 7(1): 3696, 2017 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-28623291

RESUMO

Myelin-gene Regulatory Factor (MyRF) is one of the master transcription factors controlling myelin formation and development in oligodendrocytes which is crucial for the powerful brain functions. The N-terminal of MyRF, which contains a proline-rich region and a DNA binding domain (DBD), is auto-cleaved from the ER membrane, and then enters the nucleus to participate in transcription regulation of the myelin genes. Here we report the crystal structure of MyRF DBD. It shows an Ig-fold like architecture which consists of two antiparallel ß-sheets with 7 main strands, packing against each other, forming a ß-sandwich. Compared to its homolog, Ndt80, MyRF has a smaller and less complex DBD lacking the helices and the big loops outside the core. Structural alignment reveals that MyRF DBD possess less interaction sites with DNA than Ndt80 and may bind only at the major groove of DNA. Moreover, the structure reveals a trimeric assembly, agreeing with the previous report that MyRF DBD functions as a trimer. The mutant that we designed based on the structure disturbed trimer formation, but didn't affect the auto-cleavage reaction. It demonstrates that the activation of self-cleavage reaction of MyRF is independent of the presence of its N-terminal DBD homotrimer. The structure reported here will help to understand the molecular mechanism underlying the important roles of MyRF in myelin formation and development.


Assuntos
DNA/química , Modelos Moleculares , Domínios e Motivos de Interação entre Proteínas , Relação Quantitativa Estrutura-Atividade , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , DNA/metabolismo , Camundongos , Conformação Molecular , Mutação , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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