Regulation of phosphoribosyl ubiquitination by a calmodulin-dependent glutamylase.

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.

Structural basis for the dual catalytic activity of the Legionella pneumophila ovarian tumor (OTU) domain deubiquitinase LotA.

Legionella pneumophila, a bacterial pathogen that causes a severe pneumonia known as Legionnaires' disease, extensively exploits the ubiquitin (Ub) pathway in the infected host cells through certain virulence effectors excreted by the Dot/Icm system. To date, several Dot/Icm effectors have been found to act as Ub ligases, and four effectors, including LotA, LotB, LotC, and Ceg7, have been identified as deubiquitinases (DUBs) from the ovarian tumor (OTU) domain family. LotA is unique among other OTU DUBs because it possesses two distinct DUB domains and exclusively exhibits catalytic activity against K6-linked diUb and polyUb chains. However, the structure of LotA and the molecular mechanism for the dual DUB activity remains elusive. In this study, we solved the structure of LotA in complex with proximally bound Ub and distal covalently bound Ub. Both Ub molecules are bound to the DUB1 domain and mimic a K6-linked diUb. Structural analysis reveals that the DUB1 domain utilizes a distinct mechanism for recognition of the K6-linked diUb within a large S1' binding site that is uncommon to OTU DUBs. Structural fold of the LotA DUB2 domain closely resembles LotB and LotC, similarly containing an extra α-helix lobe that has been demonstrated to play an important role in Ub binding. Collectively, our study uncovers the structural basis for the dual catalytic activity of the unique OTU family DUB LotA.

Structural and functional insights into a novel two-component endolysin encoded by a single gene in Enterococcus faecalis phage.

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.

Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system.

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.

The bacterial deubiquitinase Ceg23 regulates the association of Lys-63-linked polyubiquitin molecules on the Legionella phagosome.

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.

Structural and biochemical analyses of the tetrameric cell binding domain of Lys170 from enterococcal phage F170/08.

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.

Structural basis of antiphage immunity generated by a prokaryotic Argonaute-associated SPARSA system.

Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD+)-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD+ binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD+ hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.

Molecular mechanism of toxin neutralization in the HipBST toxin-antitoxin system of Legionella pneumophila.

Toxin-antitoxin (TA) systems are ubiquitous genetic modules in bacteria and archaea. Here, we perform structural and biochemical characterization of the Legionella pneumophila effector Lpg2370, demonstrating that it is a Ser/Thr kinase. Together with two upstream genes, lpg2370 constitutes the tripartite HipBST TA. Notably, the toxin Lpg2370 (HipTLp) and the antitoxin Lpg2369 (HipSLp) correspond to the C-terminus and N-terminus of HipA from HipBA TA, respectively. By determining crystal structures of autophosphorylated HipTLp, its complex with AMP-PNP, and the structure of HipTLp-HipSLp complex, we identify residues in HipTLp critical for ATP binding and those contributing to its interactions with HipSLp. Structural analysis reveals that HipSLp binding induces a loop-to-helix shift in the P-loop of HipTLp, leading to the blockage of ATP binding and inhibition of the kinase activity. These findings establish the L. pneumophila effector Lpg2370 as the HipBST TA toxin and elucidate the molecular basis for HipT neutralization in HipBST TA.

A secreted effector with a dual role as a toxin and as a transcriptional factor.

Bacteria have evolved multiple secretion systems for delivering effector proteins into the cytosol of neighboring cells, but the roles of many of these effectors remain unknown. Here, we show that Yersinia pseudotuberculosis secretes an effector, CccR, that can act both as a toxin and as a transcriptional factor. The effector is secreted by a type VI secretion system (T6SS) and can enter nearby cells of the same species and other species (such as Escherichia coli) via cell-cell contact and in a contact-independent manner. CccR contains an N-terminal FIC domain and a C-terminal DNA-binding domain. In Y. pseudotuberculosis cells, CccR inhibits its own expression by binding through its DNA-binding domain to the cccR promoter, and affects the expression of other genes through unclear mechanisms. In E. coli cells, the FIC domain of CccR AMPylates the cell division protein FtsZ, inducing cell filamentation and growth arrest. Thus, our results indicate that CccR has a dual role, modulating gene expression in neighboring cells of the same species, and inhibiting the growth of competitors.

Crystal structure of the MyRF ICA domain with its upstream ß-helical stalk reveals the molecular mechanisms underlying its trimerization and self-cleavage.

Myelin gene regulatory factor (MyRF), a novel membrane transcription factor expressed on the endoplasmic reticulum membrane, functions as a trimer. The trimerization of MyRF is associated with a fragment between the DNA binding domain and transmembrane domain that shares homology with the triple-ß-helix and intramolecular chaperone autocleavage (ICA) domain of phage tailspike proteins. The molecular details of these domains in eukaryotes have not been elucidated. Here, we present the crystal structure of the MyRF ICA domain with its upstream ß-helical stalk, determined at 2.4Å resolution. The structure showed that its upstream ß-helical stalk is different from the triple ß-helix reported before. This is the first structure of the mammalian protein with a triple ß-helix. Structure analysis demonstrated that the triple α-helical coiled-coil formed at the MyRF ICA domain C-terminal was the main driving force for the trimerization. Additionally, our findings showed that MyRF was cleaved via a highly conserved serine-lysine catalytic dyad mechanism and that cleavage would be activated only if the ICA domains were organized as trimers. In contrast to the viral ICA domain, almost no interaction was found between the MyRF ICA domain and its upstream neighboring ß-helix of the stalk; thus, activation of self-cleavage may not be triggered by the upstream region of the ICA domain, contrary to the observations made in phages. These findings provided an important insight into the molecular mechanisms of MyRF trimerization and self-cleavage.

Interplay between bacterial deubiquitinase and ubiquitin E3 ligase regulates ubiquitin dynamics on Legionella phagosomes.

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.

DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis.

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.

DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis. Corrigendum.

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

Crystal structure of the DNA-binding domain of Myelin-gene Regulatory Factor.

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.