Crystal structure of Staphylococcus aureus lipase complex with unsaturated petroselinic acid.

Staphylococcus aureus produces large amounts of toxins and virulence factors. In patients with underlying diseases or compromised immune systems, this bacterium can lead to severe infections and potentially death. In this study, the crystal structure of the complex of S. aureus lipase (SAL), which is involved in the growth of this bacterium, with petroselinic acid (PSA), an inhibitor of unsaturated fatty acids, was determined by X-ray crystallography. Recently, PSA was shown to inhibit S. aureus biofilm formation and the enzymatic activity of SAL. To further characterize the inhibitory mechanism, we determined the half-inhibitory concentration of SAL by PSA and the crystal structure of the complex. The IC50 of the inhibitory effect of PSA on SAL was 3.4 µm. SAL and PSA inhibitors were co-crystallized, and diffraction data sets were collected to 2.19 Å resolution at SPring-8 to determine the crystal structure and elucidate the detailed structural interactions. The results show that the fatty acid moiety of PSA is tightly bound to a hydrophobic pocket extending in two directions around the catalytic residue Ser116. Ser116 was also covalently bonded to the carbon of the unsaturated fatty acid moiety, and an oxyanion hole in SAL stabilized the electrons of the double bond. The difference in inhibitory activity between PSA and ester compounds revealed a structure-activity relationship between SAL and PSA. Additional research is required to further characterize the clinical potential of PSA.

Correction: Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0204160.].

Crystal structure of pathogenic Staphylococcus aureus lipase complex with the anti-obesity drug orlistat.

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor and may be a therapeutic target for infectious diseases. Herein, we determined the 3D structure of native SAL, the mutated S116A inactive form, and the inhibitor complex using the anti-obesity drug orlistat to aid in drug development. The determined crystal structures showed a typical α/ß hydrolase motif with a dimeric form. Fatty acids bound near the active site in native SAL and inactive S116A mutant structures. We found that orlistat potently inhibits SAL activity, and it covalently bound to the catalytic Ser116 residue. This is the first report detailing orlistat-lipase binding. It provides structure-based information on the production of potent anti-SAL drugs and lipase inhibitors. These results also indicated that orlistat can be repositioned to treat bacterial diseases.

Structural insights into the unique polylactate-degrading mechanism of Thermobifida alba cutinase.

Cutinases are enzymes known to degrade polyester-type plastics. Est119, a plastic-degrading type of cutinase from Thermobifida alba AHK119 (herein called Ta_cut), shows a broad substrate specificity toward polyesters, and can degrade substrates including polylactic acid (PLA). However, the PLA-degrading mechanism of cutinases is still poorly understood. Here, we report the structure complexes of cutinase with ethyl lactate (EL), the constitutional unit. From this complex structure, the electron density maps clearly showed one lactate (LAC) and one EL occupying different positions in the active site cleft. The binding mode of EL is assumed to show a figure prior to reaction and LAC is an after-reaction product. These complex structures demonstrate the role of active site residues in the esterase reaction and substrate recognition. The complex structures were compared with other documented complex structures of cutinases and with the structure of PETase from Ideonella sakaiensis. The amino acid residues involved in substrate interaction are highly conserved among these enzymes. Thus, mapping the precise interactions in the Ta_cut and EL complex will pave the way for understanding the plastic-degrading mechanism of cutinases and suggest ways of creating more potent enzymes by structural protein engineering.

Significance of a histone-like protein with its native structure for the diagnosis of asymptomatic tuberculosis.

Tuberculosis causes the highest mortality among all single infections. Asymptomatic tuberculosis, afflicting one third of the global human population, is the major source as 5-10% of asymptomatic cases develop active tuberculosis during their lifetime. Thus it is one of important issues to develop diagnostic tools for accurately detecting asymptomatic infection. Mycobacterial DNA-binding protein 1 (MDP1) is a major protein in persistent Mycobacterium tuberculosis and has potential for diagnostic use in detecting asymptomatic infection. However, a previous ELISA-based study revealed a specificity problem; IgGs against MDP1 were detected in both M. tuberculosis-infected and uninfected individuals. Although the tertiary structures of an antigen are known to influence antibody recognition, the MDP1 structural details have not yet been investigated. The N-terminal half of MDP1, homologous to bacterial histone-like protein HU, is predicted to be responsible for DNA-binding, while the C-terminal half is assumed as totally intrinsically disordered regions. To clarify the relationship between the MDP1 tertiary structure and IgG recognition, we refined the purification method, which allow us to obtain a recombinant protein with the predicted structure. Furthermore, we showed that an IgG-ELISA using MDP1 purified by our refined method is indeed useful in the detection of asymptomatic tuberculosis.

Staphylococcus aureus lipase: purification, kinetic characterization, crystallization and crystallographic study.

Staphylococcus aureus lipase (SAL), a triacylglycerol esterase, is an important virulence factor in S. aureus and may be a therapeutic target for infectious diseases caused by S. aureus. For the purposes of anti-SAL drug development using structure-based drug design, X-ray crystallographic analysis of SAL overexpressed in Escherichia coli was performed. The recombinant protein was purified using a three-step protocol involving immobilized metal-affinity chromatography, cation-exchange chromatography and anion-exchange chromatography flowthrough methods, yielding 40 mg of protein per litre of bacterial culture. Crystals were obtained using the sitting-drop vapor-diffusion technique. Diffraction data to 3.0 Šresolution were collected on the BL44XU beamline at SPring-8 at the zinc peak of 1.2842 Šfor SAD phasing. The crystals belonged to space group P4122 or P4322, with unit-cell parameters a = 131.0, b = 131.0, c = 250.6 Å, and are likely to contain four SAL molecules (408 residues) per asymmetric unit.

Crystal structure of Clostridium botulinum whole hemagglutinin reveals a huge triskelion-shaped molecular complex.

Clostridium botulinum HA is a component of the large botulinum neurotoxin complex and is critical for its oral toxicity. HA plays multiple roles in toxin penetration in the gastrointestinal tract, including protection from the digestive environment, binding to the intestinal mucosal surface, and disruption of the epithelial barrier. At least two properties of HA contribute to these roles: the sugar-binding activity and the barrier-disrupting activity that depends on E-cadherin binding of HA. HA consists of three different proteins, HA1, HA2, and HA3, whose structures have been partially solved and are made up mainly of ß-strands. Here, we demonstrate structural and functional reconstitution of whole HA and present the complete structure of HA of serotype B determined by x-ray crystallography at 3.5 Å resolution. This structure reveals whole HA to be a huge triskelion-shaped molecule. Our results suggest that whole HA is functionally and structurally separable into two parts: HA1, involved in recognition of cell-surface carbohydrates, and HA2-HA3, involved in paracellular barrier disruption by E-cadherin binding.

Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium.

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.

Crystallization and preliminary crystallographic studies of the HA3 subcomponent of the type B botulinum neurotoxin complex.

The haemagglutinin subcomponent HA3 of the type B botulinum neurotoxin complex, which is important in toxin absorption from the gastrointestinal tract, has been expressed, purified and subsequently crystallized in two crystal forms at different pH values. Form I belonged to space group R32, with unit-cell parameters a = b = 357.4, c = 249.5 Å, α = ß = 90, γ = 120°. Form II belonged to space group I4(1)32, with unit-cell parameters a = b = c = 259.0 Å, α = ß = γ = 90°. Diffraction data were collected from these crystals to a resolution of 3.0 Å for both form I and form II.

A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+) into Fe(3+) and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1), a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m) values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c), Mycobacterium tuberculosis (Rv2986c), and Mycobacterium leprae (ML1683; ML-LBP) were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

Enzymatic actions of Pasteurella multocida toxin detected by monoclonal antibodies recognizing the deamidated α subunit of the heterotrimeric GTPase Gq.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some Pasteurellosis. PMT exerts its toxic effects through the activation of heterotrimeric GTPase (G(q), G(12/13) and G(i))-dependent pathways, by deamidating a glutamine residue in the α subunit of these GTPases. However, the enzymatic characteristics of PMT are yet to be analyzed in detail because the deamidation has only been observed in cell-based assays. In the present study, we developed rat monoclonal antibodies, specifically recognizing the deamidated Gα(q), to detect the actions of PMT by immunological techniques such as western blotting. Using the monoclonal antibodies, we found that the toxin deamidated Gα(q) only under reducing conditions. The C-terminal region of PMT, C-PMT, was more active than the full-length PMT. The C3 domain possessing the enzyme core catalyzed the deamidation in vitro without any other domains. These results not only support previous observations on toxicity, but also provide insights into the enzymatic nature of PMT. In addition, we present several lines of evidence that Gα(11), as well as Gα(q), could be a substrate for PMT.

Crystal structure of Clostridium perfringens enterotoxin displays features of beta-pore-forming toxins.

Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of ß-pore-forming toxins such as aerolysin, C. perfringens ε-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of ß-strands, two of which span its entire length. Domain II and domain III have three short ß-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the ß-strands. The sequence of amino acids composing the α-helix and preceding ß-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming ß-barrel-made pores. These structural features imply that CPE is a ß-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long ß-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins.

Characterization of the membrane-targeting C1 domain in Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT) is a virulence factor responsible for the pathogenesis of some forms of pasteurellosis. The toxin activates G(q)- and G(12/13)-dependent pathways through the deamidation of a glutamine residue in the alpha-subunit of heterotrimeric GTPases. We recently reported the crystal structure of the C terminus (residues 575-1285) of PMT (C-PMT), which is composed of three domains (C1, C2, and C3), and that the C1 domain is involved in the localization of C-PMT to the plasma membrane, and the C3 domain possesses a cysteine protease-like catalytic triad. In this study, we analyzed the membrane-targeting function of the C1 domain in detail. The C1 domain consists of seven helices of which the first four (residues 590-670), showing structural similarity to the N terminus of Clostridium difficile toxin B, were found to be involved in the recruitment of C-PMT to the plasma membrane. C-PMT lacking these helices (C-PMT DeltaC1(4H)) neither localized to the plasma membrane nor stimulated the G(q/12/13)-dependent signaling pathways. When the membrane-targeting property was complemented by a peptide tag with an N-myristoylation motif, C-PMT DeltaC1(4H) recovered the PMT activity. Direct binding between the C1 domain and liposomes containing phospholipids was evidenced by surface plasmon resonance analyses. These results indicate that the C1 domain of C-PMT functions as a targeting signal for the plasma membrane.

Human Nei-like protein NEIL3 has AP lyase activity specific for single-stranded DNA and confers oxidative stress resistance in Escherichia coli mutant.

Oxidative base damage leads to alteration of genomic information and is implicated as a cause of aging and carcinogenesis. To combat oxidative damage to DNA, cells contain several DNA glycosylases including OGG1, NTH1 and the Nei-like proteins, NEIL1 and NEIL2. A third Nei-like protein, NEIL3, is composed of an amino-terminal Nei-like domain and an unknown carboxy-terminal domain. In contrast to the other well-described DNA glycosylases, the DNA glycosylase activity and in vivo repair function of NEIL3 remains unclear. We show here that the structural modeling of the putative NEIL3 glycosylase domain (1-290) fits well to the known Escherichia coli Fpg crystal structure. In spite of the structural similarity, the recombinant NEIL3 and NEIL3(1-290) proteins do not cleave any of several test oligonucleotides containing a single modified base. Within the substrates, we detected AP lyase activity for single-stranded (ss) DNA but double-stranded (ds) DNA. The activity is abrogated completely in mutants with an amino-terminal deletion and at the zinc-finger motif. Surprisingly, NEIL3 partially rescues an E. coli nth nei mutant from hydrogen peroxide sensitivity. Taken together, repair of certain base damage including base loss in ssDNA may be mediated by NEIL3.

Crystal structures reveal a thiol protease-like catalytic triad in the C-terminal region of Pasteurella multocida toxin.

Pasteurella multocida toxin (PMT), one of the virulence factors produced by the bacteria, exerts its toxicity by up-regulating various signaling cascades downstream of the heterotrimeric GTPases Gq and G12/13 in an unknown fashion. Here, we present the crystal structure of the C-terminal region (residues 575-1,285) of PMT, which carries an intracellularly active moiety. The overall structure of C-terminal region of PMT displays a Trojan horse-like shape, composed of three domains with a "feet"-,"body"-, and "head"-type arrangement, which were designated C1, C2, and C3 from the N to the C terminus, respectively. The C1 domain, showing marked similarity in steric structure to the N-terminal domain of Clostridium difficile toxin B, was found to lead the toxin molecule to the plasma membrane. The C3 domain possesses the Cys-His-Asp catalytic triad that is organized only when the Cys is released from a disulfide bond. The steric alignment of the triad corresponded well to that of papain or other enzymes carrying Cys-His-Asp. PMT toxicities on target cells were completely abrogated when one of the amino acids constituting the triad was mutated. Our results indicate that PMT is an enzyme toxin carrying the cysteine protease-like catalytic triad dependent on the redox state and functions on the cytoplasmic face of the plasma membrane of target cells.

Crystallization and preliminary crystallographic studies of the Pasteurella multocida toxin catalytic domain.

The C-terminal catalytic domain of Pasteurella multocida toxin, which is the virulence factor of the organism in P. multocida, has been expressed, purified and subsequently crystallized using the sitting-drop vapour-diffusion technique. Native diffraction data to 1.9 A resolution were obtained at the BL44XU beamline of SPring-8 from a flash-frozen crystal at 100 K. The crystals belong to space group C2, with unit-cell parameters a = 111.0, b = 150.4, c = 77.1 A, beta = 105.5 degrees, and are likely to contain one C-PMT (726 residues) per asymmetric unit.

[Structural biology of human CD81, a receptor for hepatitis C virus].

Human CD81, which is belonged to tetraspanin family, has been previously identified as a receptor for the hepatitis C virus envelope E 2 glycoprotein. The crystal structure of the human CD81 long extracellular domain, binding site for E 2 glycoprotein, is presented here at 1.6 A resolution. The tertiary structure of CD81-LEL, which is composed of five alpha-helices, is resemble for a mushroom-shaped molecules (stalk and head subdomains) and forms a dimer in the crystallographic asymmetric unit. The two disulfide bridges, which are conserved all the tetraspanin and are necessary for CD 81-HCV interaction, are stabilizing the conformation of the head domain. This head domain is solvent exposed surface region and is locating the amino acid residues which are essential for the E 2 binding. The hydrophobic cluster in this head domain may suggest that the presence of a docking site for a low complementary surface cavity in the partner E 2 glycoprotein. We proposed that the dimer structure may be important in the interactions of HCV E 2 glycoprotein and also the viral protein may occur in dimeric aggregation on the HCV envelope. This common structural motif of the tetraspanin provides the first insight onto the mechanism of HCV binding to human cell and may be targets for structure-based antiviral drug.

Subunit association and conformational flexibility in the head subdomain of human CD81 large extracellular loop.

The large extracellular loop of human CD81, a tetraspanin mediating hepatitis C virus envelope protein E2 binding to human cells, has been crystallized in a hexagonal form. The three-dimensional structure, solved and refined at 2.6 A resolution (R-factor = 22.8%), shows that the protein adopts a dimeric assembly, based on an association interface built up by tetraspanin-conserved residues. Structural comparisons with the tertiary structure of human CD81 large extracellular loop, previously determined in a different crystal form, show marked conformational fluctuations in the molecular regions thought to be involved in binding to the viral protein, suggesting rules for recognition and assembly within the tetraspan web.