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d -Alanine- d -alanine ligase (DDL) catalyzes the biosynthesis of d -alanyl- d -alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 A. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d -alanyl- d -alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that in-house Zn-SAD phasing can be widely applicable to structure determination.

EstU1 is a unique family VIII carboxylesterase that displays hydrolytic activity toward the amide bond of clinically used β-lactam antibiotics as well as the ester bond of p-nitrophenyl esters. EstU1 assumes a β-lactamase-like modular architecture and contains the residues Ser100, Lys103, and Tyr218, which correspond to the three catalytic residues (Ser64, Lys67, and Tyr150, respectively) of class C β-lactamases. The structure of the EstU1/cephalothin complex demonstrates that the active site of EstU1 is not ideally tailored to perform an efficient deacylation reaction during the hydrolysis of β-lactam antibiotics. This result explains the weak β-lactamase activity of EstU1 compared with class C β-lactamases. Finally, structural and sequential comparison of EstU1 with other family VIII carboxylesterases elucidates an operative molecular strategy used by family VIII carboxylesterases to extend their substrate spectrum.

The N-terminal DNA-binding domain of BldD from Streptomyces coelicolor A3(2) was crystallized by the hanging-drop vapour-diffusion method at 296 K. A 1.8 angstroms data set has been collected using synchrotron radiation at Pohang Light Source, South Korea. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 77.2, b = 31.8, c = 33.6 angstroms, beta = 105.1 degrees. Analysis of the packing density shows that the asymmetric unit probably contains one molecule, with a solvent content of 43.6%.

Plasmid-encoded class C beta-lactamases, including CMY-1 and CMY-10, hydrolyze the lactam bonds of beta-lactam antibiotics, inducing therapeutic failure and a lack of eradication of clinical isolates by third-generation cephalosporins or cephamycins. Therefore, the enzymes are potential targets for developing agents against pathogens isolated from patients suffering from wound infection, urinary tract infection or pneumonia. CMY-1 and CMY-10 were purified and crystallized at 298 K. X-ray diffraction data from CMY-1 and CMY-10 crystals have been collected to 2.5 and 1.5 A resolution, respectively, using synchrotron radiation. The crystals of the two proteins are isomorphous and belong to the primitive monoclinic space group P2(1).

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism., Author Summary Legionella pneumophila is a pathogen bacterium that causes Legionnaires' disease accompanied by severe pneumonia. Surprisingly, this pathogen invades and replicates inside macrophages, whose major function is to detect and destroy invading microorganisms. How L. pneumophila can be “immune” to this primary immune cell has been a focus of intensive research. Upon being engulfed by a macrophage cell, L. pneumophila translocates hundreds of bacterial proteins into this host cell. These proteins, called bacterial effectors, are thought to manipulate normal host cellular processes. However, which host molecules and how they are targeted by the bacterial effectors are largely unknown. In this study, we determined the three-dimensional structure of L. pneumophila effector protein VipD, whose function in macrophage was unknown. Ensuing analyses revealed that VipD selectively and tightly binds two host signaling proteins Rab5 and Rab22, which are key regulators of early endosomal vesicle trafficking. These interactions prevent the activated form of Rab5 and Rab22 from binding their downstream signaling proteins, resulting in the blockade of endosomal trafficking in macrophages. The presented work shows that L. pneumophila targets endosomal Rab proteins and delineates the underlying molecular mechanism, providing a new insight into the pathogen's strategies to dysregulate normal intracellular processes.

Communication between osteoblasts and endothelial cells is essential for bone fracture repair, but the molecular identities of such communicating factors are not well defined. Here we identify DJ-1 as a novel mediator of the cross-talk between osteoblasts and endothelial cells through an unbiased screening of molecules secreted from human mesenchymal stem cells during osteogenesis. We show that DJ-1 stimulates the differentiation of human mesenchymal stem cells to osteoblasts and that DJ-1 induces angiogenesis in endothelial cells through activation of fibroblast growth factor receptor-1 signalling. In a rodent model of bone fracture repair, extracellular application of DJ-1 enhances bone regeneration in vivo by stimulating the formation of blood vessels and new bones. Both these effects are blocked by antagonizing fibroblast growth factor receptor-1 signalling. These findings uncover previously undefined extracellular roles of DJ-1 to promote angiogenesis and osteogenesis, suggesting DJ-1 may have therapeutic potential to stimulate bone regeneration.

International audience; YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner, and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation, scrambled RNase isomerization and insulin reduction, and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL-affinity column, separated by nonreducing-reducing SDS-PAGE and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxydative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity and metabolic deficiencies.

Zinc is one of the essential transition metals in cells. Excess or lack of zinc is detrimental, and cells exploit highly sensitive zinc-binding regulators to achieve homeostasis. In this article, we present a crystal structure of active Zur from Streptomyces coelicolor with three zinc-binding sites (C-, M-, and D-sites). Mutations of the three sites differentially affected sporulation and transcription of target genes, such that C- and M-site mutations inhibited sporulation and derepressed all target genes examined, whereas D-site mutations did not affect sporulation and derepressed only a sensitive gene. Biochemical and spectroscopic analyses of representative metal site mutants revealed that the C-site serves a structural role, whereas the M- and D-sites regulate DNA-binding activity as an on-off switch and a fine-tuner, respectively. Consistent with differential effect of mutations on target genes, zinc chelation by TPEN derepressed some genes ( znuA, rpmF2 ) more sensitively than others ( rpmG2 , SCO7682) in vivo. Similar pattern of TPEN-sensitivity was observed for Zur-DNA complexes formed on different promoters in vitro. The sensitive promoters bound Zur with lower affinity than the less sensitive ones. EDTA-treated apo-Zur gained its DNA binding activity at different concentrations of added zinc for the two promoter groups, corresponding to free zinc concentrations of 4.5 × 10 −16 M and 7.9 × 10 −16 M for the less sensitive and sensitive promoters, respectively. The graded expression of target genes is a clever outcome of subtly modulating Zur-DNA binding affinities in response to zinc availability. It enables bacteria to detect metal depletion with improved sensitivity and optimize gene-expression pattern.

CTX-M-15, an extended-spectrum β-lactamase emerging worldwide, hydrolyzes lactam ring of β-lactam antibiotics, and thus causes therapeutic failure and a lack of eradication of pathogenic bacteria by third-generation β-lactams. Therefore, the enzyme is a potential target for developing agents against pathogens isolated from patients suffering from nosocomial infections. The CTX-M-15 protein was purified and crystallized at 298 K. X-ray diffraction data from CTX-M-15 crystal have been collected to 1.46 Å resolution using synchrotron radiation. The crystal of CTX-M-15 belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.50, b = 44.23, and c = 116.92 Å. Analysis of the packing density shows that the asymmetric unit probably contains two molecules with a solvent content of 41.26%.

The enormous pool of biodiversity in marine ecosystems is an excellent natural reservoir for acquiring an inventory of enzymes with potential for biotechnological applications. Moreover, the opportunity for sustainable resource management has been greatly enhanced by recent advances in culturing methods for recalcitrant microbes. In this review, we will focus primarily on successful examples in culturing marine microbes and provide an overview of work examining the biotechnological potential of the marine reservoir, mainly through genomic strategies, such as activity-based functional screening of genomic and metagenomic libraries and homology-driven screening of enormous amounts of sequence data.

Lon is an oligomeric ATP-dependent protease that degrades defective or denatured proteins as well as some folded proteins for the control of cellular protein quality and metabolism. Lon from Thermococcus onnurineus NA1 was purified and crystallized at 295 K. A 2.0 A resolution data set was collected using synchrotron radiation. The crystals belonged to space group P6(3), with unit-cell parameters a = 121.45, b = 121.45, c = 195.24 A. Assuming the presence of two monomers in the asymmetric unit, the solvent content was estimated to be about 60.7%.

Nur, a member of the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and anti-oxidative response in Streptomyces coelicolor. Here we report the 2.4-A resolution crystal structure of Nur. It contains a unique nickel-specific metal site in addition to a nonspecific common metal site. The identification of the 6-5-6 motif of the Nur recognition box and a Nur/DNA complex model reveals that Nur mainly interacts with terminal bases of the palindrome on complex formation. This contrasts with more distributed contacts between Fur and the n-1-n type of the Fur-binding motif. The disparity between Nur and Fur in the conformation of the S1-S2 sheet in the DNA-binding domain can explain their different DNA-recognition patterns. Furthermore, the fact that the specificity of Nur in metal sensing and DNA recognition is conferred by the specific metal site suggests that its introduction drives the evolution of Nur orthologs in the Fur family.

Gram-negative bacteria-producing extended-spectrum β-lactamases (ESBLs) are found to be truly multiresistant pathogens causing severe clinical problems. In our investigations, fifteen class C β-lactamases with extended substrate spectra have been reported in Gram-negative pathogens. Because of the emergence and dissemination of these enzymes, we propose that these enzymes be recognized as class C ESBLs (cESBLs), although most of the known ESBLs are class A and D β-lactamases. To decrease the selective pressure of antimicrobial drugs and minimize antimicrobial resistance, it is necessary for health-care professionals to recognize the presence of emerging cESBLs as a new and disturbing trend in antimicrobial resistance of Gram-negative pathogens. Because there is currently no drug development against cESBL-producing Gram-negative pathogens in progress and large pharmaceutical companies have largely withdrawn from research and development of new antimicrobial drugs, there is a tremendous need for the development of new β-lactams (or β-lactamase inhibitors) by focused cooperation between academia and small pharmaceutical companies, using the similar structural mechanism (a potential therapeutic target) of the extended substrate spectrum shown in most cESBLs. The consensus view about antimicrobial resistance is that severe clinical problems arise from the emergence of antibiotic resistance in Gram-negative pathogens causing nosocomial infections, and from the lack of new antimicrobial agents to challenge the threat [1]. There are four disturbing trends (extending substrate spectra) in the increasing antimicrobial resistance of Gram-negative pathogens [1]: (i) class B β-lactamases (metallo-β-lactamases) conferring resistance to almost all β-lactam antibiotics [2]; (ii) a bifunctional aminoglycoside-modifying enzyme [3]; (iii) the evolution of a fluoroquinolone-modifying enzyme from an aminoglycoside acetyltransferase [4]; and (iv) a new plasmid-borne fluoroquinolone efflux determinant [5]. These disturbing trends indicate that options for the treatment of health-care–associated Gram-negative infections are perilously limited as the organisms expand their ability to evade existing antimicrobial agents [1],[6]. Here we wish to draw attention to a new disturbing trend (the recently emerging class C extended-spectrum β-lactamases [ESBLs]), and to the antimicrobial drug development for class C ESBLs. We suggest also that the category of ESBLs has to be expanded.

The TON_0887 gene product from Thermococcus onnurineus NA1 is a 240-residue protein that has histidinol-phosphate phosphatase (HolPase) activity. According to analysis of its primary structure, the TON_0887 gene product is a monofunctional HolPase that belongs to the DDDD superfamily. This contrasts with the generally accepted classification that bifunctional HolPases belong to the DDDD superfamily. The TON_0887 gene product was purified and crystallized at 295 K. A 2.2 A resolution data set was collected using synchrotron radiation. The TON-HolPase crystals belonged to space group P222(1), with unit-cell parameters a = 40.88, b = 46.89, c = 148.03 A. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 48.3%.

Mutations in the DJ-1 gene have been implicated in the autosomal recessive early onset parkinsonism. DJ-1 is a soluble dimeric protein with critical roles in response to oxidative stress and in neuronal maintenance. However, several lines of evidence suggest the existence of a nonfunctional aggregated form of DJ-1 in the brain of patients with some neurodegenerative diseases. Here, we show that inorganic phosphate, an important anion that exhibits elevated levels in patients with Parkinson disease, transforms DJ-1 into filamentous aggregates. According to the 2.4-Å crystal structure, DJ-1 dimers are linearly stacked through Pi-mediated interactions to form protofilaments, which are then bundled into a filamentous assembly.

Members of the genus Thermococcus , sulfur-reducing hyperthermophilic archaea, are ubiquitously present in various deep-sea hydrothermal vent systems and are considered to play a significant role in the microbial consortia. We present the complete genome sequence and feature analysis of Thermococcus onnurineus NA1 isolated from a deep-sea hydrothermal vent area, which reveal clues to its physiology. Based on results of genomic analysis, T. onnurineus NA1 possesses the metabolic pathways for organotrophic growth on peptides, amino acids, or sugars. More interesting was the discovery that the genome encoded unique proteins that are involved in carboxydotrophy to generate energy by oxidation of CO to CO 2 , thereby providing a mechanistic basis for growth with CO as a substrate. This lithotrophic feature in combination with carbon fixation via RuBisCO (ribulose 1,5-bisphosphate carboxylase/oxygenase) introduces a new strategy with a complementing energy supply for T. onnurineus NA1 potentially allowing it to cope with nutrient stress in the surrounding of hydrothermal vents, providing the first genomic evidence for the carboxydotrophy in Thermococcus .

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes pro-mutagenic uracils in the DNA template, leading to stalling of DNA polymerases. Here, we describe our new findings regarding the molecular mechanism underpinning the stalling of polymerases. We observed that two successive deaminated bases were required to stall TNA1 and KOD1 DNA polymerases, whereas a single deaminated base was enough for stalling Pfu DNA polymerase, in spite of the virtually identical uracil-sensing domains. TNA1 and KOD1 DNA polymerases have a much higher extension rate than Pfu DNA polymerase; decreasing the extension rate resulted in stalling by TNA1 and KOD1 DNA polymerases at a single deaminated base. These results strongly suggest that these polymerases require two factors to stop DNA polymerization at a single deaminated base: the presence of the uracil-sensing domain and a relatively slow extension rate.

Human karyopherin beta3, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin beta3 complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin beta3 and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin beta3 and apo A-I. In addition, overexpression of karyopherin beta3 significantly increased apo A-I secretion. These results suggest that karyopherin beta3 plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin beta3 and coronary artery diseases associated with apo A-I.

In Escherichia coli , glucose-dependent transcriptional induction of genes encoding a variety of sugar-metabolizing enzymes and transport systems is mediated by the phosphorylation state-dependent interaction of membrane-bound enzyme IICB Glc (EIICB Glc ) with the global repressor Mlc. Here we report the crystal structure of a tetrameric Mlc in a complex with four molecules of enzyme IIB Glc (EIIB), the cytoplasmic domain of EIICB Glc . Each monomer of Mlc has one bound EIIB molecule, indicating the 1:1 stoichiometry. The detailed view of the interface, along with the high-resolution structure of EIIB containing a sulfate ion at the phosphorylation site, suggests that the phosphorylation-induced steric hindrance and disturbance of polar intermolecular interactions impede complex formation. Furthermore, we reveal that Mlc possesses a built-in flexibility for the structural adaptation to its target DNA and that interaction of Mlc with EIIB fused only to dimeric proteins resulted in the loss of its DNA binding ability, suggesting that flexibility of the Mlc structure is indispensable for its DNA binding.

Escherichia coli QOR2 [NAD(P)H-dependent quinone oxidoreductase; a ytfG gene product], which catalyzes two-electron reduction of methyl-1,4-benzoquinone, is a new type of quinone-reducing enzyme with distinct primary sequence and oligomeric conformation from previously known quinone oxidoreductases. The crystal structures of native QOR2 and the QOR2-NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) complex reveal that QOR2 consists of two domains (N-domain and C-domain) resembling those of NmrA, a negative transcriptional regulator that belongs to the short-chain dehydrogenase/reductase family. The N-domain, which adopts the Rossmann fold, provides a platform for NADPH binding, whereas the C-domain, which contains a hydrophobic pocket connected to the NADPH-binding site, appears to play important roles in substrate binding. Asn143 near the NADPH-binding site has been identified to be involved in substrate binding and catalysis from structural and mutational analyses. Moreover, compared with wild-type strain, the qor2-overexpressing strain shows growth retardation and remarkable decrease in several enzymes involved in carbon metabolism, suggesting that QOR2 could play some physiological roles in addition to quinone reduction.

Nickel ions serve in the correct folding and function of microbial enzymes implicated in metabolic processes. Although nickel ions are indispensable for the survival of cells, the intracellular level of nickel ions needs to be properly maintained as excessive levels of nickel ions are toxic. Nur, a nickel-uptake regulator belonging to the Fur family, is a nickel-responsive transcription factor that controls nickel homeostasis and antioxidative response in Streptomyces coelicolor. Nur was purified and crystallized at 295 K. A 2.4 A native data set and a 3.0 A Ni-MAD data set were collected using synchrotron radiation. The Nur crystals belong to space group P3(1), with unit-cell parameters a = b = 78.17, c = 50.39 A. Assuming the presence of two molecules in the asymmetric unit, the solvent content is estimated to be about 54.7%.

The B30.2/SPRY domain is present in approximately 700 eukaryotic (approximately 150 human) proteins, including medically important proteins such as TRIM5alpha and Pyrin. Nonetheless, the functional role of this modular domain remained unclear. Here, we report the crystal structure of an SPRY-SOCS box family protein GUSTAVUS in complex with Elongins B and C, revealing a highly distorted two-layered beta-sandwich core structure of its B30.2/SPRY domain. Ensuing studies identified one end of the beta-sandwich as the surface interacting with an RNA helicase VASA with a 40 nM dissociation constant. The sequence variation in TRIM5alpha responsible for HIV-1 restriction and most of the mutations in Pyrin causing familial Mediterranean fever map on this surface, implicating the corresponding region in many B30.2/SPRY domains as the ligand-binding site. The amino acids lining the binding surface are highly variable among the B30.2/SPRY domains, suggesting that these domains are protein-interacting modules, which recognize a specific individual partner protein rather than a consensus sequence motif.

KSI (ketosteroid isomerase) catalyses an allylic isomerization reaction at a diffusion-controlled rate. A hydrogen bond network, Asp(99).Water(504).Tyr(14).Tyr(55).Tyr(30), connects two critical catalytic residues, Tyr(14) and Asp(99), with Tyr(30), Tyr(55) and a water molecule in the highly apolar active site of the Pseudomonas putida KSI. In order to characterize the interactions among these amino acids in the hydrogen bond network of KSI, double-mutant cycle analysis was performed, and the crystal structure of each mutant protein within the cycle was determined respectively to interpret the coupling energy. The DeltaDeltaG(o) values of Y14F/D99L (Tyr(14)-->Phe/Asp(99)-->Leu) KSI, 25.5 kJ/mol for catalysis and 28.9 kJ/mol for stability, were smaller than the sums (i.e. 29.7 kJ/mol for catalysis and 34.3 kJ/mol for stability) for single mutant KSIs respectively, indicating that the effect of the Y14F/D99L mutation was partially additive for both catalysis and stability. The partially additive effect of the Y14F/D99L mutation suggests that Tyr(14) and Asp(99) should interact positively for the stabilization of the transition state during the catalysis. The crystal structure of Y14F/D99L KSI indicated that the Y14F/D99L mutation increased the hydrophobic interaction while disrupting the hydrogen bond network. The DeltaDeltaG(o) values of both Y30F/D99L and Y55F/D99L KSIs for the catalysis and stability were larger than the sum of single mutants, suggesting that either Tyr(30) and Asp(99) or Tyr(55) and Asp(99) should interact negatively for the catalysis and stability. These synergistic effects of both Y30F/D99L and Y55F/D99L mutations resulted from the disruption of the hydrogen bond network. The synergistic effect of the Y55F/D99L mutation was larger than that of the Y30F/D99L mutation, since the former mutation impaired the proper positioning of a critical catalytic residue, Tyr(14), involved in the catalysis of KSI. The present study can provide insight into interpreting the coupling energy measured by double-mutant cycle analysis based on the crystal structures of the wild-type and mutant proteins.

TRAIL is a member of the tumor necrosis factor (TNF) superfamily. TRAIL has drawn a lasting attention because of its selectivity and efficacy in inducing apoptosis in a variety of cancer cells but not in normal cells. The structures of both TRAIL and the protein in complex with the extracellular domain of death receptor 5 (sDR5) were elucidated. Because each factor of the ligand family and the receptor family is large, it poses an intriguing question of how recognition between cognate ligands and receptors is achieved in a highly specific manner without cross interactions. This review focuses on the unique properties of TRAIL and molecular strategies for the specific recognition between the two family members primarily based on the crystal structures of TRAIL and the TRAIL:sDR5 complex.

TRAIL is a member of the tumor necrosis factor (TNF) superfamily. TRAIL has drawn a lasting attention because of its selectivity and efficacy in inducing apoptosis in a variety of cancer cells but not in normal cells. The structures of both TRAIL and the protein in complex with the extracellular domain of death receptor 5 (sDR5) were elucidated. Because each factor of the ligand family and the receptor family is large, it poses an intriguing question of how recognition between cognate ligands and receptors is achieved in a highly specific manner without cross interactions. This review focuses on the unique properties of TRAIL and molecular strategies for the specific recognition between the two family members primarily based on the crystal structures of TRAIL and the TRAIL:sDR5 complex.

The Escherichia coli ytfG gene product, with NAD(P)H:quinone oxidoreductase activity, was crystallized by the hanging-drop vapour-diffusion method at 296 K. A 1.78 A data set has been collected using synchrotron radiation at Pohang Light Source, South Korea. The crystal belongs to the primitive trigonal system, with unit-cell parameters a = b = 81.7, c = 76.8 A. Analysis of the packing density shows that the asymmetric unit probably contains one monomer, with a solvent content of 48.8%.

A yedU gene product with a molecular mass of 31 kDa is a hypothetical protein with no known function. The protein was purified and crystallized at 296 K. X-ray diffraction data have been collected to 2.3 A using synchrotron radiation. The crystals belong to the primitive orthorhombic system, with unit-cell parameters a = 50.56, b = 63.45, c = 168.02 A. The asymmetric unit contains two monomers of the protein, with a corresponding V(M) of 2.25 A(3) Da(-1) and a solvent content of 44.84%.

A sequence alignment shows that residue 332 is conserved as glutamate in maltogenic amylases (MAases) and in other related enzymes such as cyclodextrinase and neopullulanase, while the corresponding position is conserved as histidine in alpha-amylases. We analyzed the role of Glu332 in the hydrolysis and the transglycosylation activity of Thermus MAase (ThMA) by site-directed mutagenesis. Replacing Glu332 with histidine reduced transglycosylation activity significantly, but enhanced hydrolysis activity on alpha-(1,3)-, alpha-(1,4)-, and alpha-(1,6)-glycosidic bonds relative to the wild-type (WT) enzyme. The mutant Glu332Asp had catalytic properties similar to those of the WT enzyme, but the mutant Glu332Gln resulted in significantly decreased transglycosylation activity. These results suggest that an acidic side chain at position 332 of MAase plays an important role in the formation and accumulation of transfer products by modulating the relative rates of hydrolysis and transglycosylation. From the structure, we propose that an acidic side chain at position 332, which is located in a pocket, is involved in aligning the acceptor molecule to compete with water molecules in the nucleophilic attack of the glycosyl-enzyme intermediate.

Amylases catalyze the hydrolysis of starch material and play central roles in carbohydrate metabolism. Compared with many different amylases that are able to hydrolyze only alpha-D-(1,4)-glycosidic bonds, maltogenic amylases exhibit catalytic versatility: hydrolysis of alpha-D-(1,4)- and alpha-D-(1,6)-glycosidic bonds and transglycosylation of oligosaccharides to C3-, C4-, or C6-hydroxyl groups of various acceptor mono- or disaccharides. It has been speculated that the catalytic property of the enzymes is linked to the additional approximately 130 residues at the N terminus that are absent in other typical alpha-amylases. The crystal structure of a maltogenic amylase from a Thermus strain was determined at 2.8 A. The structure, an analytical centrifugation, and a size exclusion column chromatography proved that the enzyme is a dimer in solution. The N-terminal segment of the enzyme folds into a distinct domain and comprises the enzyme active site together with the central (alpha/beta)(8) barrel of the adjacent subunit. The active site is a narrow and deep cleft suitable for binding cyclodextrins, which are the preferred substrates to other starch materials. At the bottom of the active site cleft, an extra space, absent in the other typical alpha-amylases, is present whose size is comparable with that of a disaccharide. The space is most likely to host an acceptor molecule for the transglycosylation and to allow binding of a branched oligosaccharide for hydrolysis of alpha-D-(1,4)-glycosidic or alpha-D-(1,6)-glycosidic bond. The (alpha/beta)(8) barrel of the enzyme is the preserved scaffold in all the known amylases. The structure represents a novel example of how an enzyme acquires a different substrate profile and a catalytic versatility from a common active site and represents a framework for explaining the catalytic activities of transglycosylation and hydrolysis of alpha-D-(1,6)-glycosidic bond.

In a recently published article (9), Wachino et al. reported that a single amino acid substitution (I292S) responsible for the expansion of the hydrolyzing activity against several extended-spectrum cephalosporins (ceftazidime or cefepime) is near the H-10 helix region of CMY-19. However, other reports (2, 7, 8) have concluded that the region responsible for the extended substrate spectrum in class C β-lactamases is in (not near) the H-10 helix. Wachino and colleagues designated the amino acid sequence from 279 to 287 as the H-10 helix (Fig. ​(Fig.1),1), which may be based on the crystal structure of AmpC K-12 (PDB code 2BLS), although they did not state the fact. However, one previous report described residues 279 to 294 as the H-10 helix, according to the crystal structure of GC1 (3). Our recent report (5) showed that the sequence from 289 to 294 was the H-10 (α10) helix (Fig. ​(Fig.1),1), based on superposed crystal structures among CMY-10 (5), P99, and GC1 β-lactamases. There are different positions of the H-10 helix that is related to the extended substrate spectrum in class C β-lactamases. Therefore, we propose that the exact region responsible for the extended substrate spectrum is the R2 loop (residues 289 to 307) (Fig. ​(Fig.1)1) described in our recent report (5). There are three important reasons why the exact region is the R2 loop in the R2 active site, referring to the region that accommodates the R2 side chain at C3 of the β-lactam nucleus in extended-spectrum cephalosporins. FIG. 1. A sequence alignment of amino acid residues near the H-9 (α9) and H-10 helix (α10) of class C β-lactamases with extended substrate spectrum. Alignment among CMY-10, P99, and GC1 β-lactamases whose structures are available ... First, the R2 loop includes all regions responsible for the extended substrate spectrum in all reported class C extended-spectrum β-lactamases except HKY28 (4): (i) six-amino-acid deletion (residues 289 to 294) of CHE (2); (ii) the single amino acid substitution (L296H) of AmpC KL (7); (iii) four-amino-acid deletion (residues 293 to 296) of HD (8); (iv) three-amino-acid deletion (residues 303 to 305) of CMY-10 (5); (v) the single amino acid substitution (I292S) of CMY-19 (9); and (vi) the L293P substitution of Ear2 (1). Second, mutations in the R2 loop can change the architecture of the active site in class C extended-spectrum β-lactamases, thereby affecting their hydrolyzing activity. Owing to the deletion in CMY-10, for example, the R2 loop in the R2 active site displays noticeable structural alterations: the shortened path of the connection R2 loop between α10 and β11 (Fig. ​(Fig.1)1) induces a ∼2.5-A shift of α9 and α10 relative to the adjacent helix α11 in CMY-10, compared with both P99 and GC1 β-lactamases, opening the gap between α9-α10 and α11 (5). Therefore, the bulky R2 side chain of extended-spectrum cephalosporins could fit snugly into the significantly widened R2 active site in this way (6). Third, CMY-19 showed 97% sequence identity to CMY-10. But the sequence identity between CMY-19 and AmpC K-12 was 40%. Therefore, it is reasonable for the region responsible for the extended substrate spectrum in CMY-19 to be designated based on the crystal structure of CMY-10 (not AmpC K-12).

The NS3 protein of hepatitis C virus (HCV) is thought to be essential for viral replication. The N-terminal domain of the protein contains protease activity and the C-terminal domain contains nucleotide triphosphatase and RNA helicase activity. The RNA helicase domain of HCV NS3 protein was purified by using affinity-column chromatographic methods, and crystallized by using the microbatch crystallization method under oil at 277 K. The crystals belong to primitive trigonal space group P3121 or P3221 with cell dimensions of a = b = 93.3, c = 104.6 Å. The asymmetric unit contains one molecule of the helicase domain, with the crystal volume per protein mass (V m ) of 2.50 Å3 Da−1 and solvent content of about 50.8% by volume. A native data set to 2.3 Å resolution was obtained from a frozen crystal indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.

A novel maltogenic amylase from Bacillus stearothermophilus ET1, which has a dual activity of α-1,4- and α-1,6-glycosidic bond cleavages and α-1,6-glycosidic bond formation, was crystallized by using the hanging-drop vapor-diffusion method. The best crystals were obtained by employing a high concentration of protein (56 mg ml−1) and a precipitant containing 22% glycerol, 1.6 M ammonium sulfate in 0.1 M Tris–HCl (pH 8.5). Native diffraction data to 2.66 Å resolution have been obtained from crystals flash-frozen at 110 K. The crystals belong to the space group P212121 with unit-cell dimensions of a = 77.62, b = 121.23, c = 244.29 Å, and contain three or four protomers per asymmetric unit. Structure determination by multiple isomorphous replacement is in progress.