ABSTRACT
Enteropathogenic E. coli NleB and related type III effectors catalyze arginine GlcNAcylation of death domain (DD) proteins to block host defense, but the underlying mechanism is unknown. Here we solve crystal structures of NleB alone and in complex with FADD-DD, UDP, and Mn2+ as well as NleB-GlcNAcylated DDs of TRADD and RIPK1. NleB adopts a GT-A fold with a unique helix-pair insertion to hold FADD-DD; the interface contacts explain the selectivity of NleB for certain DDs. The acceptor arginine is fixed into a cleft, in which Glu253 serves as a base to activate the guanidinium. Analyses of the enzyme-substrate complex and the product structures reveal an inverting sugar-transfer reaction and a detailed catalytic mechanism. These structural insights are validated by mutagenesis analyses of NleB-mediated GlcNAcylation in vitro and its function in mouse infection. Our study builds a structural framework for understanding of NleB-catalyzed arginine GlcNAcylation of host death domain.
Subject(s)
Enteropathogenic Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Host-Pathogen Interactions/genetics , Protein Conformation , Virulence Factors/chemistry , Animals , Apoptosis/genetics , Arginine/chemistry , Arginine/genetics , Coenzyme A Ligases/chemistry , Coenzyme A Ligases/genetics , Crystallography, X-Ray , Death Domain/genetics , Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Guanidine/chemistry , Humans , Manganese/chemistry , Mice , Mutagenesis , TNF Receptor-Associated Death Domain Protein/chemistry , TNF Receptor-Associated Death Domain Protein/genetics , Virulence Factors/geneticsABSTRACT
Immune recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors often activates proinflammatory NF-κB signalling1. Recent studies indicate that the bacterial metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (HBP) can activate NF-κB signalling in host cytosol2-4, but it is unclear whether HBP is a genuine PAMP and the cognate pattern recognition receptor has not been identified. Here we combined a transposon screen in Yersinia pseudotuberculosis with biochemical analyses and identified ADP-ß-D-manno-heptose (ADP-Hep), which mediates type III secretion system-dependent NF-κB activation and cytokine expression. ADP-Hep, but not other heptose metabolites, could enter host cytosol to activate NF-κB. A CRISPR-Cas9 screen showed that activation of NF-κB by ADP-Hep involves an ALPK1 (alpha-kinase 1)-TIFA (TRAF-interacting protein with forkhead-associated domain) axis. ADP-Hep directly binds the N-terminal domain of ALPK1, stimulating its kinase domain to phosphorylate and activate TIFA. The crystal structure of the N-terminal domain of ALPK1 and ADP-Hep in complex revealed the atomic mechanism of this ligand-receptor recognition process. HBP was transformed by host adenylyltransferases into ADP-heptose 7-P, which could activate ALPK1 to a lesser extent than ADP-Hep. ADP-Hep (but not HBP) alone or during bacterial infection induced Alpk1-dependent inflammation in mice. Our findings identify ALPK1 and ADP-Hep as a pattern recognition receptor and an effective immunomodulator, respectively.
Subject(s)
Adenosine Diphosphate Sugars/immunology , Burkholderia cenocepacia , Cytosol , Immunity, Innate , Pathogen-Associated Molecular Pattern Molecules/immunology , Protein Kinases/metabolism , Yersinia pseudotuberculosis , Adenosine Diphosphate Sugars/metabolism , Animals , Burkholderia Infections/enzymology , Burkholderia Infections/immunology , Burkholderia Infections/pathology , Burkholderia cenocepacia/genetics , Burkholderia cenocepacia/immunology , Burkholderia cenocepacia/metabolism , CRISPR-Cas Systems , Crystallography, X-Ray , Cytokines/biosynthesis , Cytosol/enzymology , Cytosol/immunology , Disaccharides/metabolism , Enzyme Activation , Female , Gene Editing , Immunologic Factors/immunology , Immunologic Factors/metabolism , Immunomodulation , Inflammation/enzymology , Inflammation/immunology , Inflammation/pathology , Male , Mice , Mice, Inbred C57BL , Models, Molecular , NF-kappa B/metabolism , Pathogen-Associated Molecular Pattern Molecules/metabolism , Yersinia pseudotuberculosis/genetics , Yersinia pseudotuberculosis/immunology , Yersinia pseudotuberculosis/metabolismABSTRACT
OBJECTIVE: Diagnosis of lupus nephritis (LN) is a complex process, which usually requires renal biopsy. We aim to establish a machine learning pipeline to help diagnosis of LN. METHODS: A cohort of 681 systemic lupus erythematosus (SLE) patients without LN and 786 SLE patients with LN was established, and a total of 95 clinical, laboratory data and 17 meteorological indicators were collected. After tenfold cross-validation, the patients were divided into training set and test set. The features selected by collective feature selection method of mutual information (MI) and multisurf were used to construct the models of logistic regression, decision tree, random forest, naive Bayes, support vector machine (SVM), light gradient boosting (LGB), extreme gradient boosting (XGB), and artificial neural network (ANN), the models were compared and verified in post-analysis. RESULTS: Collective feature selection method screens out antistreptolysin (ASO), retinol binding protein (RBP), lupus anticoagulant 1 (LA1), LA2, proteinuria and other features, and the hyperparameter optimized XGB (ROC: AUC = 0.995; PRC: AUC = 1.000, APS = 1.000; balance accuracy: 0.990) has the best performance, followed by LGB (ROC: AUC = 0.992; PRC: AUC = 0.997, APS = 0.977; balance accuracy: 0.957). The worst performance is naive Bayes model (ROC: AUC = 0.799; PRC: AUC = 0.822, APS = 0.823; balance accuracy: 0.693). In the composite feature importance bar plots, ASO, RF, Up/Ucr, and other features play important roles in LN. CONCLUSION: We developed and validated a new and simple machine learning pathway for diagnosis of LN, especially the XGB model based on ASO, LA1, LA2, proteinuria, and other features screened out by collective feature selection.
Subject(s)
Lupus Erythematosus, Systemic , Lupus Nephritis , Humans , Lupus Nephritis/diagnosis , Bayes Theorem , Proteinuria , Machine LearningABSTRACT
OBJECTIVE: Clinical evaluation of systemic lupus erythematosus (SLE) disease activity is limited and inconsistent, and high disease activity significantly, seriously impacts on SLE patients. This study aims to generate a machine learning model to identify SLE patients with high disease activity. METHOD: A total of 1014 SLE patients with low disease activity and 453 SLE patients with high disease activity were included. A total of 94 clinical, laboratory data and 17 meteorological indicators were collected. After data preprocessing, we use mutual information and multisurf to evaluate and select the importance of features. The selected features are used for machine learning modeling. Performance of the model is evaluated and verified by a series of binary classification indicators. RESULTS: We screened out hematuria, proteinuria, pyuria, low complement, precipitation, sunlight and other features for model construction by integrated feature selection. After hyperparameter optimization, the LGB has the best performance (ROC: AUC = 0.930; PRC: AUC = 0.911, APS = 0.913; balance accuracy: 0.856), and the worst is the naive bayes (ROC: AUC = 0.849; PRC: AUC = 0.719, APS = 0.714; balance accuracy: 0.705). Finally, the selection of features has good consistency in the composite feature importance bar plot. CONCLUSION: We identify SLE patients with high disease activity by a simple machine learning pipeline, especially the LGB model based on the characteristics of proteinuria, hematuria, pyuria and other feathers screened out by collective feature selection.
Subject(s)
Lupus Erythematosus, Systemic , Pyuria , Humans , Hematuria , Bayes Theorem , Lupus Erythematosus, Systemic/diagnosis , Machine Learning , ProteinuriaABSTRACT
Inflammatory caspases cleave the gasdermin D (GSDMD) protein to trigger pyroptosis, a lytic form of cell death that is crucial for immune defences and diseases. GSDMD contains a functionally important gasdermin-N domain that is shared in the gasdermin family. The functional mechanism of action of gasdermin proteins is unknown. Here we show that the gasdermin-N domains of the gasdermin proteins GSDMD, GSDMA3 and GSDMA can bind membrane lipids, phosphoinositides and cardiolipin, and exhibit membrane-disrupting cytotoxicity in mammalian cells and artificially transformed bacteria. Gasdermin-N moved to the plasma membrane during pyroptosis. Purified gasdermin-N efficiently lysed phosphoinositide/cardiolipin-containing liposomes and formed pores on membranes made of artificial or natural phospholipid mixtures. Most gasdermin pores had an inner diameter of 1014 nm and contained 16 symmetric protomers. The crystal structure of GSDMA3 showed an autoinhibited two-domain architecture that is conserved in the gasdermin family. Structure-guided mutagenesis demonstrated that the liposome-leakage and pore-forming activities of the gasdermin-N domain are required for pyroptosis. These findings reveal the mechanism for pyroptosis and provide insights into the roles of the gasdermin family in necrosis, immunity and diseases.
Subject(s)
Cell Membrane Permeability/drug effects , Proteins/antagonists & inhibitors , Proteins/chemistry , Pyroptosis , Animals , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , Cardiolipins/metabolism , Caspases/metabolism , Cell Line , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Crystallography, X-Ray , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Liposomes , Membrane Lipids/metabolism , Mice , Models, Molecular , Necrosis , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/metabolism , Phosphate-Binding Proteins , Phosphatidylinositols/metabolism , Porosity/drug effects , Protein Structure, Tertiary , Proteins/metabolism , Proteins/pharmacology , Pyroptosis/drug effects , Pyroptosis/immunologyABSTRACT
The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain-containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.
Subject(s)
Bacterial Proteins/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/ultrastructure , Escherichia coli/genetics , Escherichia coli/ultrastructure , Escherichia coli Proteins/genetics , Escherichia coli Proteins/ultrastructure , Flagella/chemistry , Flagella/genetics , Flagella/ultrastructure , Gene Expression Regulation, Bacterial , Guanosine Monophosphate/chemistry , Protein Binding/geneticsABSTRACT
Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Comamonas testosteroni/metabolism , Methyl-Accepting Chemotaxis Proteins/chemistry , Methyl-Accepting Chemotaxis Proteins/metabolism , Bacterial Proteins/genetics , Chemotaxis , Citric Acid/metabolism , Comamonas testosteroni/chemistry , Comamonas testosteroni/genetics , Dimerization , Ligands , Methyl-Accepting Chemotaxis Proteins/genetics , Protein Binding , Protein Conformation , Protein Conformation, alpha-Helical , Protein DomainsABSTRACT
Phaeosphaeria fuckelii, an endophytic fungus associated with the herbal medicine Phlomis umbrosa, produced four new thiodiketopiperazine alkaloids, phaeosphaones A-D (1-4), featuring an unusual ß-(oxy)thiotryptophan motif, along with four known analogues, phaeosphaone E (5), chetoseminudin B (6), polanrazine B (7), and leptosin D (8). Their structures were elucidated by extensive spectroscopic data analysis, and their absolute configurations were determined by single-crystal X-ray diffraction and ECD calculations. Compounds 4, 6, and 8 were found to display mushroom tyrosinase inhibitory activity with IC50 values of 33.2 ± 0.2, 31.7 ± 0.2, and 28.4 ± 0.2 µM, respectively, more potent than that of the positive control, kojic acid (IC50 = 40.4 ± 0.1 µM). A molecular-docking study disclosed the π-π stacking interaction between the indole moiety of 8 and the His243 residue of tyrosinase.
Subject(s)
Alkaloids/chemistry , Ascomycota/chemistry , Monophenol Monooxygenase/antagonists & inhibitors , Agaricales , Crystallography, X-Ray , Molecular StructureABSTRACT
L,D-transpeptidases, widely distributed in bacteria and even in the difficult-to-treat ESKAPE pathogens, can confer antibacterial resistance against the traditional ß-lactam antibiotics through bypass of the 4â¯ââ¯3 transpeptide linkage. LdtMt2, a l,d-transpeptidase in Mycobacteria tuberculosis, is essential for bacterial virulence and is considered as a potential anti-tuberculosis target inhibited by carbapenems. Diverse interaction modes between carbapenems and LdtMt2 have been reported, there are only limited evidences to validate those interaction modes. Herein, we identified the stable binding states of two carbapenems, imipenem and ertapenem, via crystallographic and biochemical studies, discovered that they adopt similar binding conformations. We further demonstrate the absence of the 1-ß-methyl group in imipenem and the presence of both Y308 and Y318 residues in LdtMt2 synergistically resulted in one order of magnitude higher affinity for imipenem than ertapenem. Our study provides a structural basis for the rational drug design and evolvement of novel carbapenems against bacterial L,D-transpeptidases.
Subject(s)
Anti-Bacterial Agents/chemistry , Ertapenem/chemistry , Imipenem/chemistry , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/enzymology , Peptidyl Transferases/chemistry , Binding Sites , Crystallography, X-Ray , Drug Design , Kinetics , Mass Spectrometry , Protein Binding , Protein ConformationABSTRACT
Drought stress is a major obstacle to agriculture. Although many studies have reported on plant drought tolerance achieved via genetic modification, application of plant growth-promoting rhizobacteria (PGPR) to achieve tolerance has rarely been studied. In this study, the ability of three isolates, including Bacillus amyloliquefaciens 54, from 30 potential PGPR to induce drought tolerance in tomato plants was examined via greenhouse screening. The results indicated that B. amyloliquefaciens 54 significantly enhanced drought tolerance by increasing survival rate, relative water content and root vigor. Coordinated changes were also observed in cellular defense responses, including decreased concentration of malondialdehyde and elevated concentration of antioxidant enzyme activities. Moreover, expression levels of stress-responsive genes, such as lea, tdi65, and ltpg2, increased in B. amyloliquefaciens 54-treated plants. In addition, B. amyloliquefaciens 54 induced stomatal closure through an abscisic acid-regulated pathway. Furthermore, we constructed biofilm formation mutants and determined the role of biofilm formation in B. amyloliquefaciens 54-induced drought tolerance. The results showed that biofilm-forming ability was positively correlated with plant root colonization. Moreover, plants inoculated with hyper-robust biofilm (ΔabrB and ΔywcC) mutants were better able to resist drought stress, while defective biofilm (ΔepsA-O and ΔtasA) mutants were more vulnerable to drought stress. Taken altogether, these results suggest that biofilm formation is crucial to B. amyloliquefaciens 54 root colonization and drought tolerance in tomato plants.
Subject(s)
Adaptation, Biological , Bacillus amyloliquefaciens/physiology , Biofilms , Droughts , Solanum lycopersicum/microbiology , Solanum lycopersicum/physiology , Stress, Physiological , Antioxidants/metabolism , Plant Development , Plant Roots/microbiology , Plant Roots/physiology , SymbiosisABSTRACT
The equilibrium between C2'- and C3'-endo conformations of nucleotides in solution, as well as their polymers DNA and RNA, has been well studied in previous work. However, this equilibrium of nucleotides in their binding state remains unclear. We observed two AMP molecules, in C3'- and C2'-endo conformations respectively, simultaneously bound to a cystathionine-beta-synthase (CBS) domain dimer of the magnesium and cobalt efflux protein CorC in the crystallographic study. The C2'-endo AMP molecule assumes the higher sugar pucker energy and one more hydrogen bond with the protein than the C3'-endo molecule does. The balance between the high sugar pucker energy and the low binding energy suggests an equilibrium or switch between C2'- and C3'-endo conformations of the bound nucleotides. Our work challenge the previous hypothesis that the ribose of the bound nucleotides would be locked in a fixed conformation.
Subject(s)
Adenosine Monophosphate/metabolism , Cobalt/chemistry , Cystathionine beta-Synthase/metabolism , Escherichia coli/metabolism , Magnesium/chemistry , Metalloproteins/metabolism , Adenosine Monophosphate/chemistry , Crystallography, X-Ray , Cystathionine beta-Synthase/chemistry , Escherichia coli/chemistry , Metalloproteins/chemistry , Molecular Conformation , Molecular Docking Simulation , Protein Binding , Protein DomainsABSTRACT
Biofilm dispersal is characterized by the cell detachment from biofilms and expected to provide novel "anti-biofilm" approaches of prevention and treatment of biofilms in clinical and industrial settings. The E.coli protein BdcA has been identified as a biofilm dispersal factor and designed to be an important component in engineered applications to control biofilm formation. It belongs to short-chain dehydrogenase/reductase (SDR) family with the specific affinity to NADPH. Here, we show the structure of BdcA in complex with NADPH and confirm that NADPH binding is requisite for BdcA facilitating cell motility and increasing biofilm dispersal. Especially, we observe a potential substrate binding pocket surrounded by hydrophobic residues upon NADPH binding and present evidences that this pocket is essential for BdcA binding NADPH and exerting its biological functions. Our study provides the clues for illuminating the molecular mechanism of BdcA regulating biofilm dispersal and better utilizing BdcA to eliminate the biofilms.
Subject(s)
Biofilms , Escherichia coli Proteins/metabolism , Escherichia coli/physiology , NADP/metabolism , Binding Sites , Crystallography, X-Ray , Escherichia coli/chemistry , Escherichia coli/cytology , Escherichia coli Proteins/chemistry , Molecular Docking Simulation , NADP/chemistry , Protein Conformation , Substrate SpecificityABSTRACT
A series of acetophenone derivatives (10a-10i, 11, 12a-12g, 13a-13g, 14a-14d and 15a-15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure-activity relationship was summarized. Compounds 10a-10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC50 values of 6.0-22.6⯵g/mL, especially Cytospora sp. (IC50â¯=â¯6.0⯵g/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.
Subject(s)
Acetophenones/pharmacology , Ascomycota/drug effects , Biological Products/pharmacology , Botrytis/drug effects , Fungicides, Industrial/pharmacology , Magnaporthe/drug effects , Acetophenones/chemical synthesis , Acetophenones/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , Dose-Response Relationship, Drug , Drug Design , Fungicides, Industrial/chemical synthesis , Fungicides, Industrial/chemistry , Inhibitory Concentration 50 , Microbial Sensitivity Tests , Molecular Structure , Structure-Activity RelationshipABSTRACT
Meroterpenoids isolated from guava (Psidium guajava) and Rhodomyrtus tomentosa possess special skeletons which incorporate terpenoids with phloroglucinol derivatives. Most of these meroterpenoids showed high cytotoxicity against cancer cell lines. However, their chemical diversity is very limited. Herein, we employed a biomimetic hetero-cycloaddition starting from ortho-quinone methides and an abundant natural product, ß-caryophyllene, to generate meroterpene-like compounds. Considering that the source plant has hyperglycemic functions, α-glucosidase was selected as a target for bioassay. Nine compounds were screened out for promising activities (IC50 < 15 µM), which were better than the positive controls genistein and acarbose. The best inhibitor 12 (IC50 2.73 µM) possesses two caryophyllene moieties. They represented a new type of skeleton possessing activities against α-glucosidase. The kinetic study exhibited that these inhibitors belong to a non-competitive type. All these inhibitors may provide an opportunity to develop a new class of antidiabetic agents.
Subject(s)
Glycoside Hydrolase Inhibitors/chemistry , Glycoside Hydrolase Inhibitors/pharmacology , Psidium/chemistry , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology , alpha-Glucosidases/metabolism , Biomimetics/methods , Crystallography, X-Ray , Cycloaddition Reaction/methods , Glycoside Hydrolase Inhibitors/chemical synthesis , Humans , Kinetics , Models, Molecular , Polycyclic Sesquiterpenes , Sesquiterpenes/chemical synthesisABSTRACT
Pathogenic mycobacteria transport virulence factors across their complex cell wall via a type VII secretion system (T7SS)/early secreted antigenic target-6 of kDa secretion system (ESX). ESX conserved component (Ecc) B, a core component of the T7SS architecture, is predicted to be a membrane bound protein, but little is known about its structure and function. Here, we characterize EccB1, showing that it is an ATPase with no sequence or structural homology to other ATPases located in the cell envelope of Mycobacterium tuberculosis H37Rv. We obtained the crystal structure of an EccB1-ΔN72 truncated transmembrane helix and performed modeling and ATP docking studies, showing that EccB1 likely exists as a hexamer. Sequence alignment and ATPase activity determination of EccB1 homologues indicated the presence of 3 conserved motifs in the N- and C-terminals of EccB1-ΔN72 that assemble together between 2 membrane proximal domains of the EccB1-ΔN72 monomer. Models of the EccB1 hexamer show that 2 of the conserved motifs are involved in ATPase activity and form an ATP binding pocket located on the surface of 2 adjacent molecules. Our results suggest that EccB may act as the energy provider in the transport of T7SS virulence factors and may be involved in the formation of a channel across the mycomembrane.
Subject(s)
Bacterial Proteins/metabolism , Ca(2+) Mg(2+)-ATPase/metabolism , Mycobacterium tuberculosis/metabolism , Periplasm/enzymology , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Cloning, Molecular , Crystallography, X-Ray , Molecular Sequence Data , Mutagenesis , Mycobacterium tuberculosis/enzymology , Protein Conformation , Sequence Homology, Amino Acid , Subcellular Fractions/metabolismABSTRACT
Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to 'open' the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA-ssDNA.
Subject(s)
Bacterial Proteins/chemistry , DNA, Single-Stranded/chemistry , Helicobacter pylori , Membrane Proteins/chemistry , Amino Acid Sequence , Bacterial Proteins/metabolism , Binding Sites , DNA, Single-Stranded/metabolism , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Multimerization , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Proteins of the Sac10b family are highly conserved in Archaea. Ssh10b, a member of the Sac10b family from the hyperthermophilic crenarchaeon Sulfolobus shibatae, binds to RNA in vivo. Here we show that binding by Ssh10b destabilizes RNA secondary structure. Structural analysis of Ssh10b in complex with a 25-bp RNA duplex containing local distortions reveals that Ssh10b binds the two RNA strands symmetrically as a tetramer with each dimer bound asymmetrically to a single RNA strand. Amino acid residues involved in double-stranded RNA binding are similar, but non-identical, to those in dsDNA binding. The dimer-dimer interaction mediated by the intermolecular ß-sheet appears to facilitate the destabilization of base pairing in the secondary structure of RNA. Our results suggest that proteins of the Sac10b family may play important roles in RNA transactions requiring destabilization of RNA secondary structure in Sulfolobus.
Subject(s)
Archaeal Proteins/chemistry , RNA, Archaeal/chemistry , RNA, Double-Stranded/chemistry , RNA-Binding Proteins/chemistry , Sulfolobus/chemistry , Archaeal Proteins/genetics , Archaeal Proteins/metabolism , Nucleic Acid Conformation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , RNA, Archaeal/genetics , RNA, Archaeal/metabolism , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Sulfolobus/genetics , Sulfolobus/metabolismABSTRACT
Cephalosporins constitute a large class of ß-lactam antibiotics clinically used as antimicrobial drugs. New Dehli metallo-ß-lactamase (NDM-1) poses a global threat to human health as it confers on bacterial pathogen resistance to almost all ß-lactams, including penicillins, cephalosporins, and carbapenems. Here we report the first crystal structures of NDM-1 in complex with cefuroxime and cephalexin, as well as NMR spectra monitoring cefuroxime and cefixime hydrolysis catalyzed by NDM-1. Surprisingly, cephalosporoate intermediates were captured in both crystal structures determined at 1.3 and 2.0 Å. These results provide detailed information concerning the mechanism and pathways of cephalosporin hydrolysis. We also present the crystal structure and enzyme assays of a D124N mutant, which reveals that D124 most likely plays a more structural than catalytic role.
Subject(s)
Biocatalysis , Cephalosporins/chemistry , beta-Lactamases/metabolism , Cephalosporins/metabolism , Crystallography, X-Ray , Hydrolysis , Models, Molecular , Protein Conformation , beta-Lactamases/chemistryABSTRACT
The fast estimation of chlorophyll content is significant for understanding the crops growth, monitoring the disease and insect, and assessing the yield of crops. This study gets the hyperspectral imagery data by using a self-developed multi-angular acquisition system during the different maize growth period, the reflectance of maize canopy was extracted accurately from the hyperspectral images under different view angles in the principal plane. The hot-dark-spot index (HDS) of red waveband was calculated through the analysis of simulated values by ACRM model and measured values, then this index was used to modify the vegetation index (TCARI), thus a new vegetation index (HD-TCARI) based on the multi-angular observation was proposed. Finally, the multi-angular hyperspectral imagery data was used to validate the vegetation indexes. The result showed that HD-TCARI could effectively reduce the LAI effects on the assessment of chlorophyll content. When the chlorophyll content was greater than 30 µg x cm(-2), the correlation (R2) between HD-TCARI and LAI was only 26.88%-28.72%. In addition, the HD-TCARI could resist the saturation of vegetation index during the assessment of high chlorophyll content. When the LAI varled from 1 to 6, the linear relation between HD-TCARI and chlorophyll content could be improved by 9% compared with TCARI. The ground validation of HD-TCARI by multi-angular hyperspectral image showed that the linear relation between HD-TCARI and chlorophyll content (R2 = 66.74%) was better than the TCARI (R2 = 39.92%), which indicated that HD-TCARI has good potentials for estimating the chlorophyll content.