Structural analysis of the peptidoglycan DL-endopeptidase CwlO complexed with its inhibitory protein IseA.

Peptidoglycan DL-endopeptidases locally cleave the peptide stem of peptidoglycan in the bacterial cell wall. This process facilitates bacterial growth and division by loosening the rigid peptidoglycan layer. IseA binds to the active site of multiple DL-endopeptidases and inhibits excessive peptidoglycan degradation that leads to cell lysis. To better understand how IseA inhibits DL-endopeptidase activity, we determined the crystal structure of the peptidoglycan DL-endopeptidase CwlO/IseA complex and compared it with that of the peptidoglycan DL-endopeptidase LytE/IseA complex. Structural analyses showed significant differences between the hydrophobic pocket-binding residues of the DL-endopeptidases (F361 of CwlO and W237 of LytE). Additionally, binding assays showed that the F361 mutation of CwlO to the bulkier hydrophobic residue, tryptophan, increased its binding affinity for IseA, whereas mutation to alanine reduced the affinity. These analyses revealed that the hydrophobic pocket-binding residue of DL-endopeptidases determines IseA-binding affinity and is required for substrate-mimetic inhibition by IseA.

Structural insights into the regulation of protein-arginine kinase McsB by McsA.

In gram-positive bacteria, phosphorylated arginine functions as a protein degradation signal in a similar manner as ubiquitin in eukaryotes. The protein-arginine phosphorylation is mediated by the McsAB complex, where McsB possesses kinase activity and McsA modulates McsB activity. Although mcsA and mcsB are regulated within the same operon, the role of McsA in kinase activity has not yet been clarified. In this study, we determined the molecular mechanism by which McsA regulates kinase activity. The crystal structure of the McsAB complex shows that McsA binds to the McsB kinase domain through a second zinc-coordination domain and the subsequent loop region. This binding activates McsB kinase activity by rearranging the catalytic site, preventing McsB self-assembly, and enhancing stoichiometric substrate binding. The first zinc-coordination and coiled-coil domains of McsA further activate McsB by reassembling the McsAB oligomer. These results demonstrate that McsA is the regulatory subunit for the reconstitution of the protein-arginine kinase holoenzyme. This study provides structural insight into how protein-arginine kinase directs the cellular protein degradation system.

Structural insights into the regulation of peptidoglycan DL-endopeptidases by inhibitory protein IseA.

Peptidoglycan, a physical barrier that protects bacteria from the environment, is constantly degraded and resynthesized for remodeling during cell growth and division. Because excessive or insufficient peptidoglycan hydrolysis affects bacterial homeostasis and viability, peptidoglycan degradation must be precisely regulated. In Bacillus subtilis, DL-endopeptidases play an essential role in peptidoglycan remodeling, and their activity is regulated by IseA. Here, we report the crystal structure of peptidoglycan DL-endopeptidase LytE complexed with IseA. In the crystal structure, the inhibitory loop connecting the two lobes of IseA blocks the active site of LytE by mimicking its substrate. Consistently, mutations in the inhibitory loop resulted in the loss of IseA activity. The structure also shows that conformational rearrangements in both LytE and IseA restrict access of the inhibitory loop to the LytE catalytic site. These results reveal an inhibition mechanism of peptidoglycan DL-endopeptidase in which the inhibitory protein mimics the substrate but is not degraded.

Crystal structure and biochemical analysis suggest that YjoB ATPase is a putative substrate-specific molecular chaperone.

AAA+ ATPases are ubiquitous proteins associated with most cellular processes, including DNA unwinding and protein unfolding. Their functional and structural properties are typically determined by domains and motifs added to the conserved ATPases domain. Currently, the molecular function and structure of many ATPases remain elusive. Here, we report the crystal structure and biochemical analyses of YjoB, a Bacillus subtilis AAA+ protein. The crystal structure revealed that the YjoB hexamer forms a bucket hat-shaped structure with a porous chamber. Biochemical analyses showed that YjoB prevents the aggregation of vegetative catalase KatA and gluconeogenesis-specific glyceraldehyde-3 phosphate dehydrogenase GapB but not citrate synthase, a conventional substrate. Structural and biochemical analyses further showed that the internal chamber of YjoB is necessary for inhibition of substrate aggregation. Our results suggest that YjoB, conserved in the class Bacilli, is a potential molecular chaperone acting in the starvation/stationary phases of B. subtilis growth.

Structural analysis of the regulation of blue-light receptors by GIGANTEA.

In Arabidopsis, GIGANTEA (GI), together with the blue-light receptors ZTL, LKP2, and FKF1, regulates degradation of the core clock protein TOC1 and the flowering repressor CDFs, thereby controlling circadian oscillation and flowering. Despite the significance of GI in diverse plant physiology, its molecular function is not much understood because of technical problems in protein preparation and a lack of structural information. Here, we report the purification of the GI monomer and the crystal structure of the GI/LKP2 complex. The crystal structure reveals that residues 1-813 of GI possess an elongated rigid structure formed by stacking hydrophobic α-helices and that the LOV domain of LKP2 binds to the middle region of the GI (residues 563-789). Interaction analysis further shows that LOV homodimers are converted to monomers by GI binding. Our results provide structural insights into the regulation of the circadian clock and photoperiodic flowering by GI and ZTL/LKP2/FKF1.

Crystal structure of a tandem B-box domain from Arabidopsis CONSTANS.

CONSTANS is a central protein in the regulation of photoperiodic flowering, which is expressed in response to day length and promotes the expression of Flowering Locus T. The tandem B-box domain in CONSTANS mediates interactions with various proteins to regulate the expression of Flowering Locus T. Although most plants, including Arabidopsis, have multiple B-box proteins, their B-box structures have not been elucidated. Here, we report the crystal structure of a tandem B-box domain from Arabidopsis CONSTANS. The crystal structure shows that each B-box adopts a canonical B-box fold and coordinates two zinc atoms. Furthermore, the crystal structure reveals that the B-box domain has a unique structure that distinguishes it from animal B-boxes at the monomer and dimer level.

The CAGE-MiR-181b-5p-S1PR1 Axis Regulates Anticancer Drug Resistance and Autophagy in Gastric Cancer Cells.

Cancer-associated gene (CAGE), a cancer/testis antigen, has been known to promote anticancer drug resistance. Since the underlying mechanisms of CAGE-promoted anticancer drug resistance are poorly understood, we established Anticancer drug-resistant gastric cancer cells (AGS R ) to better elucidate possible mechanisms. AGS R showed an increased expression level of CAGE and autophagic flux compared with anticancer drug-sensitive parental gastric cancer cells (AGS cells). AGS R cells showed higher invasion potential, growth rate, tumor spheroid formation, and angiogenic potential than AGS cells. CAGE exerted effects on the response to anticancer drugs and autophagic flux. CAGE was shown to bind to Beclin1, a mediator of autophagy. Overexpression of CAGE increased autophagic flux and invasion potential but inhibited the cleavage of PARP in response to anticancer drugs in CAGE CRISPR-Cas9 cell lines. TargetScan analysis was utilized to predict the binding of miR-302b-5p to the promoter sequences of CAGE, and the results show that miR-302b-5p directly regulated CAGE expression as illustrated by luciferase activity. MiR-302b-5p regulated autophagic flux and the response to anticancer drugs. CAGE was shown to bind the promoter sequences of miR-302b-5p. The culture medium of AGS R cells increased CAGE expression and autophagic flux in AGS cells. ImmunoEM showed CAGE was present in the exosomes of AGS R cells; exosomes of AGS R cells and human recombinant CAGE protein increased CAGE expression, autophagic flux, and resistance to anticancer drugs in AGS cells. MicroRNA array revealed miR-181b-5p as a potential negative regulator of CAGE. MiR-181b-5p inhibitor increased the expression of CAGE and autophagic flux in addition to preventing anticancer drugs from cleaving poly(ADP-ribose) polymerase (PARP) in AGS cells. TargetScan analysis predicted sphingosine 1-phosphate receptor 1 (SIPR1) as a potential target for miR-181b-5p. CAGE showed binding to the promoter sequences of S1PR1. The downregulation or inhibition of S1PR1 led to decreased autophagic flux but enhanced the sensitivity to anticancer drugs in AGS R cells. This study presents a novel role of the CAGE-miR-181b-5p-S1PR1 axis in anticancer drug resistance and autophagy.

Crystal structure of the DNA-binding domain of Bacillus subtilis CssR.

Bacteria utilize two-component systems to regulate gene expression in response to changes in environmental stimuli. CssS/CssR, a two-component system in Bacillus subtilis, is responsible for overcoming envelope stresses caused by heat shock and secretion overload. During stress, the sensor component CssS is auto-phosphorylated and transfers the phosphoryl group to the response regulator CssR. Phosphorylated CssR then directly regulates the transcription of genes required to counteract the stress. Here, the crystal structure of the DNA-binding domain of CssR, determined at 1.07 Å resolution, is reported. The structure shows that the DNA-binding domain of CssR harbors a winged helix-turn-helix motif that is conserved in the OmpR/PhoB subfamily of response regulators. Based on the crystal structure, the dimeric architecture of the full-length CssR and its DNA-binding mode were suggested.

Structural insights into the regulation of SigB activity by RsbV and RsbW.

Bacillus subtilis SigB is an alternative sigma factor that initiates the transcription of stress-responsive genes. The anti-sigma factor RsbW tightly binds SigB to suppress its activity under normal growth conditions and releases it when nonphosphorylated RsbV binds to RsbW in response to stress signals. To understand the regulation of SigB activity by RsbV and RsbW based on structural features, crystal structures and a small-angle X-ray scattering (SAXS) envelope structure of the RsbV-RsbW complex were determined. The crystal structures showed that RsbV and RsbW form a heterotetramer in a similar manner to a SpoIIAA-SpoIIAB tetramer. Multi-angle light scattering and SAXS revealed that the RsbV-RsbW complex is an octamer in solution. Superimposition of the crystal structure on the SAXS envelope structure showed that the unique dimeric interface of RsbW mediates the formation of an RsbV-RsbW octamer and does not prevent RsbV and SigB from binding to RsbW. These results provide structural insights into the molecular assembly of the RsbV-RsbW complex and the regulation of SigB activity.

Selective interactions between mimivirus uracil-DNA glycosylase and inhibitory proteins determined by a single amino acid.

Uracil-N-glycosylase (UNG) is found in most organisms as well as in large DNA viruses. Its inhibitory proteins, including uracil glycosylase inhibitor (UGI) and p56, tightly bind to the active site of UNG by mimicking the DNA substrates. As the binding motifs are conserved in UNG family proteins, the inhibitory proteins bind to various UNG proteins across species. However, the intercalation residue that penetrates the DNA minor groove during uracil excision is not conserved among UNG proteins. To understand the role of the intercalation residue in their binding to the inhibitory proteins, we prepared mutants of mimivirus UNG, measured the binding affinity between the UNG mutants and inhibitory proteins, and analyzed the interactions based on the crystal structures of mimivirus UNG mutants complexed with UGI. The results show that mimivirus UNG, which harbors Tyr as an intercalation residue, did not interact with the inhibitory proteins intrinsically, whereas mutations of the intercalation residue to Phe or Leu resulted in tight interactions with UGI and p56; mutation to Met resulted in tight interactions only with p56. The crystal structures revealed that Phe and Leu stabilize the interactions by fitting into the hydrophobic pocket of UGI. These results show that differences in size and hydrophobicity of the intercalation residues determine the interactions between UNG family proteins and the inhibitory proteins, UGI and p56.

Structural insights into stressosome assembly.

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA-RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA-RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Šresolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

Structural analysis of the recognition of the -35 promoter element by SigW from Bacillus subtilis.

Sigma factors are key proteins that mediate the recruitment of RNA polymerase to the promoter regions of genes, for the initiation of bacterial transcription. Multiple sigma factors in a bacterium selectively recognize their cognate promoter sequences, thereby inducing the expression of their own regulons. In this paper, we report the crystal structure of the σ4 domain of Bacillus subtilis SigW bound to the -35 promoter element. Purine-specific hydrogen bonds of the -35 promoter element with the recognition helix α9 of the σ4 domain occurs at three nucleotides of the consensus sequence (G-35, A-34, and G'-31 in G-35A-34A-33A-32C-31C-30T-29). The hydrogen bonds of the backbone with the α7 and α8 of the σ4 domain occurs at G'-30. These results elucidate the structural basis of the selective recognition of the promoter by SigW. In addition, comparison of SigW structures complexed with the -35 promoter element or with anti-sigma RsiW reveals that DNA recognition and anti-sigma factor binding of SigW are mutually exclusive.

AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

As a master regulator for metabolic and energy homeostasis, AMPK controls the activity of metabolic enzymes and transcription factors in response to cellular ATP status. AMPK has been thus recognized as a main target for the regulation of cellular energy metabolism. Here, we report that AMPK can be down-regulated by the cullin-RING ubiquitin E3 ligase 4A (CRL4A) with cereblon (CRBN). CRL4A interacted with AMPK holoenzymes and mediated AMPKα-specific polyubiquitination for its proteasomal degradation through non-K48 polyubiquitin linkages. In the ubiquitination system, CRBN was required for efficient polyubiquitination of AMPKα subunits. Consistently, polyubiquitination of AMPKα subunits was reduced by inhibitors of CRL4A-CRBN. Physiologic function of AMPK down-regulation by CRL4-CRBN was also confirmed using mouse bone marrow-derived mast cells (BMMCs). The inactivation of CRL4A-CRBN in BMMC increased AMPK stability and suppressed secretion of allergic mediators via AMPK activation followed by MAPK inhibition. In addition, CRBN knockout of BMMC also decreased allergic responses in mice. Our results suggest that the CRL4A-CRBN axis could be a target for the regulation of AMPK-dependent responses.-Kwon, E., Li, X., Deng, Y., Chang, H. W., Kim, D. Y. AMPK is down-regulated by the CRL4A-CRBN axis through the polyubiquitination of AMPKα isoforms.

Correction to: ß-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells.

In the original version of above mentioned article an error occurred in Fig. 2. Panel g and panel h are included in the figure legend, but have not been published in the figure.

ß-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells.

PURPOSE: Over half of the colon cancer patients suffer from cancer-related events, mainly metastasis. Loss of ß-catenin activity has previously been found to facilitate cancer cell dissociation and migration. Here, we aimed to investigate whether epigenetic silencing of ß-catenin induces human colon cancer cell migration and/or invasion. METHODS: HCT-116, Caco-2, HT-29 and SW620 cell migration and invasion capacities were assessed using scratch wound healing and Matrigel invasion assays, respectively. Confocal microscopy, qRT-PCR and Western blotting were performed to determine gene expression levels, whereas methylation-specific quantitative real-time PCR was used to assess the extent of ß-catenin gene (CTNNB1) promoter methylation after treatment of the cells with TPA, hydrogen peroxide, 5-aza-2'-deoxycytidine and/or VAS2870. RESULTS: We found that treatment of HT-29 and Caco-2 cells (differentiated and low metastatic) with 12-O-tetradecanoyl phorbol-13-acetate (TPA; a tumor promoter) suppressed E-cadherin and ß-catenin expression at both the mRNA and protein levels and, in addition, enhanced cell migration. Furthermore, we found that the CTNNB1 gene promoter methylation levels were higher in the more invasive HCT-116 and SW620 colon cancer cells than in HT-29 and CCD-841 (normal colon epithelial) cells. We also found that TPA or hydrogen peroxide induced CTNNB1 gene promoter methylation to a higher extent in HT-29 and CCD-841 cells than in HCT-116 and SW620 cells, and that the degree of CTNNB1 gene promoter methylation positively correlated with cell dissociation and migration. In addition, we found that co-treatment with 5-aza-2'-deoxycytidine (decitabine, a DNA methyl transferase inhibitor) and VAS2870 (a NADPH oxidase inhibitor) almost completely blocked the invasion of TPA-treated HT-29 and TPA-untreated HCT-116 and SW620 cells, and that these inhibitions surpassed those of the cells treated with decitabine or VAS2870 alone. CONCLUSIONS: From our data we conclude that the extent of CTNNB1 gene promoter methylation by reactive oxygen species correlates with the migratory and invasive abilities of colon cancer cells. Our results suggest that epigenetic regulation of CTNNB1 may serve as a novel avenue to block colon cancer cell migration and invasion.

4-Hydroxynonenal-induced GPR109A (HCA2 receptor) activation elicits bipolar responses, Gαi -mediated anti-inflammatory effects and Gßγ -mediated cell death.

BACKGROUND AND PURPOSE: In this study, we examined the possibility that 4-hydroxynonenal (4-HNE) acting as a ligand for the HCA2 receptor (GPR109A) elicits both anti-inflammatory and cell death responses. EXPERIMENTAL APPROACH: Agonistic activity of 4-HNE was determined by observing the inhibition of cAMP generation in CHO-K1-GPR109A-Gi cell line, using surface plasmon resonance (SPR) binding and competition binding assays with [3 H]-niacin. 4-HNE-mediated signalling pathways and cellular responses were investigated in cells expressing GPR109A and those not expressing these receptors. KEY RESULTS: Agonistic activity of 4-HNE was stronger than that of niacin or 3-OHBA at inhibiting forskolin-induced cAMP production and SPR binding affinity. In ARPE-19 and CCD-841 cells, activation of GPR109A by high concentrations of the agonists 4-HNE (≥10 µM), niacin (≥1000 µM) and 3-OHBA (≥1000 µM) induced apoptosis accompanied by elevated Ca2+ and superoxide levels. This 4-HNE-induced cell death was blocked by knockdown of GPR109A or NOX4 genes, or treatment with chemical inhibitors of Gßγ (gallein), intracellular Ca2+ (BAPTA-AM), NOX4 (VAS2870) and JNK (SP600125), but not by the cAMP analogue 8-CPT-cAMP. By contrast, low concentrations of 4-HNE, niacin and 3-OHBA down-regulated the expression of pro-inflammatory cytokines IL-6 and IL-8. These 4-HNE-induced inhibitory effects were blocked by a cAMP analogue but not by inhibitors of Gßγ -downstream signalling molecules. CONCLUSIONS AND IMPLICATIONS: These results revealed that 4-HNE is a strong agonist for GPR109A that induces Gαi -dependent anti-inflammatory and Gßγ -dependent cell death responses. Moreover, the findings indicate that specific intracellular signalling molecules, but not GPR109A, can serve as therapeutic targets to block 4-HNE-induced cell death.

Crystal structure of mimivirus uracil-DNA glycosylase.

Cytosine deamination induced by stresses or enzymatic catalysis converts deoxycytidine into deoxyuridine, thereby introducing a G to A mutation after DNA replication. Base-excision repair to correct uracil to cytosine is initiated by uracil-DNA glycosylase (UDG), which recognizes and eliminates uracil from DNA. Mimivirus, one of the largest known viruses, also encodes a distinctive UDG gene containing a long N-terminal domain (N-domain; residues 1-130) and a motif-I (residues 327-343), in addition to the canonical catalytic domain of family I UDGs (also called UNGs). To understand the structural and functional features of the additional segments, we have determined the crystal structure of UNG from Acanthamoeba polyphaga mimivirus (mvUNG). In the crystal structure of mvUNG, residues 95-130 in the N-domain bind to a hydrophobic groove in the catalytic domain, and motif-I forms a short ß-sheet with a positively charged surface near the active site. Circular dichroism spectra showed that residues 1-94 are in a random coil conformation. Deletion of the three additional fragments reduced the activity and thermal stability, compared to full-length mvUNG. The results suggested that the mvUNG N-domain and motif-I are required for its structural and functional integrity.

Structural insights into the regulation of Bacillus subtilis SigW activity by anti-sigma RsiW.

Bacillus subtilis SigW is localized to the cell membrane and is inactivated by the tight interaction with anti-sigma RsiW under normal growth conditions. Whereas SigW is discharged from RsiW binding and thus initiates the transcription of its regulon under diverse stress conditions such as antibiotics and alkaline shock. The release and activation of SigW in response to extracytoplasmic signals is induced by the regulated intramembrane proteolysis of RsiW. As a ZAS (Zinc-containing anti-sigma) family protein, RsiW has a CHCC zinc binding motif, which implies that its anti-sigma activity may be regulated by the state of zinc coordination in addition to the proteolytic cleavage of RsiW. To understand the regulation mode of SigW activity by RsiW, we determined the crystal structures of SigW in complex with the cytoplasmic domain of RsiW, and compared the conformation of the CHCC motif in the reduced/zinc binding and the oxidized states. The structures revealed that RsiW inhibits the promoter binding of SigW by interacting with the surface groove of SigW. The interaction between SigW and RsiW is not disrupted by the oxidation of the CHCC motif in RsiW, suggesting that SigW activity might not be regulated by the zinc coordination states of the CHCC motif.

Analysis of a draft genome sequence of Kitasatospora cheerisanensis KCTC 2395 producing bafilomycin antibiotics.

Kitasatospora cheerisanensis KCTC 2395, producing bafilomycin antibiotics belonging to plecomacrolide group, was isolated from a soil sample at Mt. Jiri, Korea. The draft genome sequence contains 8.04 Mb with 73.6% G+C content and 7,810 open reading frames. All the genes for aerial mycelium and spore formations were confirmed in this draft genome. In phylogenetic analysis of MurE proteins (UDP-N-acetylmuramyl-(L)-alanyl-(D)-glutamate:DAP ligase) in a conserved dcw (division of cell wall) locus, MurE proteins of Kitasatospora species were placed in a separate clade between MurEs of Streptomyces species incorporating (LL)-diaminopimelic acid (DAP) and MurEs of Saccharopolyspora erythraea as well as Mycobacterium tuberculosis ligating meso-DAP. From this finding, it was assumed that Kitasatospora MurEs exhibit the substrate specificity for both (LL)-DAP and meso-DAP. The bafilomycin biosynthetic gene cluster was located in the left subtelomeric region. In 71.3 kb-long gene cluster, 17 genes probably involved in the biosynthesis of bafilomycin derivatives were deduced, including 5 polyketide synthase (PKS) genes comprised of 12 PKS modules.

CBFß stabilizes HIV Vif to counteract APOBEC3 at the expense of RUNX1 target gene expression.

The HIV-1 accessory protein Vif hijacks a cellular Cullin-RING ubiquitin ligase, CRL5, to promote degradation of the APOBEC3 (A3) family of restriction factors. Recently, the cellular transcription cofactor CBFß was shown to form a complex with CRL5-Vif and to be essential for A3 degradation and viral infectivity. We now demonstrate that CBFß is required for assembling a well-ordered CRL5-Vif complex by inhibiting Vif oligomerization and by activating CRL5-Vif via direct interaction. The CRL5-Vif-CBFß holoenzyme forms a well-defined heterohexamer, indicating that Vif simultaneously hijacks CRL5 and CBFß. Heterodimers of CBFß and RUNX transcription factors contribute toward the regulation of genes, including those with immune system functions. We show that binding of Vif to CBFß is mutually exclusive with RUNX heterodimerization and impacts the expression of genes whose regulatory domains are associated with RUNX1. Our results provide a mechanism by which a pathogen with limited coding capacity uses one factor to hijack multiple host pathways.