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Find video protocols related to scientific articles indexed in Pubmed.
Structure of the gas vesicle protein GvpF from the cyanobacterium Microcystis aeruginosa.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 07-27-2014
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Gas vesicles are gas-filled proteinaceous organelles that provide buoyancy for bacteria and archaea. A gene cluster that is highly conserved in various species encodes about 8-14 proteins (Gvp proteins) that are involved in the formation of gas vesicles. Here, the first crystal structure of the gas vesicle protein GvpF from Microcystis aeruginosa PCC 7806 is reported at 2.7?Å resolution. GvpF is composed of two structurally distinct domains (the N-domain and C-domain), both of which display an ?+? class overall structure. The N-domain adopts a novel fold, whereas the C-domain has a modified ferredoxin fold with an apparent variation owing to an extension region consisting of three sequential helices. The two domains pack against each other via interactions with a C-terminal tail that is conserved among cyanobacteria. Taken together, it is concluded that the overall architecture of GvpF presents a novel fold. Moreover, it is shown that GvpF is most likely to be a structural protein that is localized at the gas-facing surface of the gas vesicle by immunoblotting and immunogold labelling-based tomography.
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Structure of pneumococcal peptidoglycan hydrolase LytB reveals insights into the bacterial cell wall remodeling and pathogenesis.
J. Biol. Chem.
PUBLISHED: 07-07-2014
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Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-?-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375-Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a "T-shaped" pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases.
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Structure of a novel O-linked N-acetyl-D-glucosamine (O-GlcNAc) transferase, GtfA, reveals insights into the glycosylation of pneumococcal serine-rich repeat adhesins.
J. Biol. Chem.
PUBLISHED: 06-16-2014
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Protein glycosylation catalyzed by the O-GlcNAc transferase (OGT) plays a critical role in various biological processes. In Streptococcus pneumoniae, the core enzyme GtfA and co-activator GtfB form an OGT complex to glycosylate the serine-rich repeat (SRR) of adhesin PsrP (pneumococcal serine-rich repeat protein), which is involved in the infection and pathogenesis. Here we report the 2.0 Å crystal structure of GtfA, revealing a ?-meander add-on domain beyond the catalytic domain. It represents a novel add-on domain, which is distinct from the all-?-tetratricopeptide repeats in the only two structure-known OGTs. Structural analyses combined with binding assays indicate that this add-on domain contributes to forming an active GtfA-GtfB complex and recognizing the acceptor protein. In addition, the in vitro glycosylation system enables us to map the O-linkages to the serine residues within the first SRR of PsrP. These findings suggest that fusion with an add-on domain might be a universal mechanism for diverse OGTs that recognize varying acceptor proteins/peptides.
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Structural insights into SraP-mediated Staphylococcus aureus adhesion to host cells.
PLoS Pathog.
PUBLISHED: 06-01-2014
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Staphylococcus aureus, a Gram-positive bacterium causes a number of devastating human diseases, such as infective endocarditis, osteomyelitis, septic arthritis and sepsis. S. aureus SraP, a surface-exposed serine-rich repeat glycoprotein (SRRP), is required for the pathogenesis of human infective endocarditis via its ligand-binding region (BR) adhering to human platelets. It remains unclear how SraP interacts with human host. Here we report the 2.05 Å crystal structure of the BR of SraP, revealing an extended rod-like architecture of four discrete modules. The N-terminal legume lectin-like module specifically binds to N-acetylneuraminic acid. The second module adopts a ?-grasp fold similar to Ig-binding proteins, whereas the last two tandem repetitive modules resemble eukaryotic cadherins but differ in calcium coordination pattern. Under the conditions tested, small-angle X-ray scattering and molecular dynamic simulation indicated that the three C-terminal modules function as a relatively rigid stem to extend the N-terminal lectin module outwards. Structure-guided mutagenesis analyses, in addition to a recently identified trisaccharide ligand of SraP, enabled us to elucidate that SraP binding to sialylated receptors promotes S. aureus adhesion to and invasion into host epithelial cells. Our findings have thus provided novel structural and functional insights into the SraP-mediated host-pathogen interaction of S. aureus.
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Piperine potentiates the hypocholesterolemic effect of curcumin in rats fed on a high fat diet.
Exp Ther Med
PUBLISHED: 03-07-2014
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It has previously been demonstrated that curcumin possesses a hypocholesterolemic effect and potentiates numerous pharmacological effects of curcumin, however, the mechanisms underlying this hypocholesterolemic effect and the interaction between curcumin and piperine remain to be elucidated. In the present study, male Sprague-Dawley rats were fed on a high-fat diet (HFD) to establish a hyperlipidemia (HLP) model. Co-administration of curcumin plus piperine was found to decrease the levels of total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol in the serum and liver, as well as increase the levels of fecal TC, TG and total bile acid, compared with administration of curcumin alone. Curcumin plus piperine also markedly increased the levels of high-density lipoprotein cholesterol. Furthermore, compared with administration of curcumin alone, administration of curcumin plus piperine resulted in a significant upregulation of the activity and gene expression of apolipoprotein AI (ApoAI), lecithin cholesterol acyltransferase (LCAT), cholesterol 7?-hydroxylase (CYP7A1) and low-density lipoprotein receptor (LDLR). In conclusion, these results indicated that co-administration of curcumin plus piperine potentiates the hypocholesterolemic effects of curcumin by increasing the activity and gene expression of ApoAI, CYP7A1, LCAT and LDLR, providing a promising combination for the treatment of HLP.
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Crystal structures and catalytic mechanism of the C-methyltransferase Coq5 provide insights into a key step of the yeast coenzyme Q synthesis pathway.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 01-23-2014
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Saccharomyces cerevisiae Coq5 is an S-adenosyl methionine (SAM)-dependent methyltransferase (SAM-MTase) that catalyzes the only C-methylation step in the coenzyme Q (CoQ) biosynthesis pathway, in which 2-methoxy-6-polyprenyl-1,4-benzoquinone (DDMQH2) is converted to 2-methoxy-5-methyl-6-polyprenyl-1,4-benzoquinone (DMQH2). Crystal structures of Coq5 were determined in the apo form (Coq5-apo) at 2.2?Å resolution and in the SAM-bound form (Coq5-SAM) at 2.4?Å resolution, representing the first pair of structures for the yeast CoQ biosynthetic enzymes. Coq5 displays a typical class I SAM-MTase structure with two minor variations beyond the core domain, both of which are considered to participate in dimerization and/or substrate recognition. Slight conformational changes at the active-site pocket were observed upon binding of SAM. Structure-based computational simulation using an analogue of DDMQH2 enabled us to identify the binding pocket and entrance tunnel of the substrate. Multiple-sequence alignment showed that the residues contributing to the dimeric interface and the SAM- and DDMQH2-binding sites are highly conserved in Coq5 and homologues from diverse species. A putative catalytic mechanism of Coq5 was proposed in which Arg201 acts as a general base to initiate catalysis with the help of a water molecule.
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Construction of a non-enzymatic glucose sensor based on copolymer P4VP-co-PAN and Fe2O3 nanoparticles.
Mater Sci Eng C Mater Biol Appl
PUBLISHED: 01-14-2014
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An electrochemical sensor based on a copolymer poly(4-vinylpyridine)-co-poly(acrylonitrile), P4VP-co-PAN, and Fe2O3 nanoparticle film modified glassy carbon electrode was developed for the determination of glucose. We studied the response of glucose with the proposed electrode, and determined the optimum conditions by changing the potential, pH and P4VP-co-PAN. The current response measurements were performed in PBS (c=0.1 M) with a potential of 0.7 V. The current response of this glucose sensor showed a linear relationship with the concentration in the range of 2.5 ?M-0.58 mM (r=0.997). The experimental results demonstrate that this method has such merits as simple operation, low cost, high sensitivity, long term stability and good reproducibility, with satisfactory results.
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Streptomyces coelicolor SCO4226 Is a Nickel Binding Protein.
PLoS ONE
PUBLISHED: 01-01-2014
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The open reading frame SCO4226 of Streptomyces coelicolor A3(2) encodes an 82-residue hypothetical protein. Biochemical assays revealed that each SCO4226 dimer binds four nickel ions. To decipher the molecular function, we solved the crystal structures of SCO4226 in both apo- and nickel-bound (Ni-SCO4226) forms at 1.30 and 2.04 Å resolution, respectively. Each subunit of SCO4226 dimer adopts a canonical ferredoxin-like fold with five ?-strands flanked by two ?-helices. In the structure of Ni-SCO4226, four nickel ions are coordinated at the surface of the dimer. Further biochemical assays suggested that the binding of Ni2+ triggers the self-aggregation of SCO4226 in vitro. In addition, RT-qPCR assays demonstrated that the expression of SCO4226 gene in S. coelicolor is specifically up-regulated by the addition of Ni2+, but not other divalent ions such as Cu2+, Mn2+ or Co2+. All these results suggested that SCO4226 acts as a nickel binding protein, probably required for nickel sequestration and/or detoxification.
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Structural and biochemical analyses of Microcystis aeruginosa O-acetylserine sulfhydrylases reveal a negative feedback regulation of cysteine biosynthesis.
Biochim. Biophys. Acta
PUBLISHED: 09-16-2013
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O-acetylserine sulfhydrylase (OASS) catalyzes the final step of cysteine biosynthesis from O-acetylserine (OAS) and inorganic sulfide in plants and bacteria. Bioinformatics analyses combined with activity assays enabled us to annotate the two putative genes of Microcystis aeruginosa PCC 7806 to CysK1 and CysK2, which encode the two 75% sequence-identical OASS paralogs. Moreover, we solved the crystal structures of CysK1 at 2.30? and cystine-complexed CysK2 at 1.91?, revealing a quite similar overall structure that belongs to the family of fold-type II PLP-dependent enzymes. Structural comparison indicated a significant induced fit upon binding to the cystine, which occupies the binding site for the substrate OAS and blocks the product release tunnel. Subsequent enzymatic assays further confirmed that cystine is a competitive inhibitor of the substrate OAS. Moreover, multiple-sequence alignment revealed that the cystine-binding residues are highly conserved in all OASS proteins, suggesting that this auto-inhibition of cystine might be a universal mechanism of cysteine biosynthesis pathway.
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Structure and catalytic mechanism of yeast 4-amino-4-deoxychorismate lyase.
J. Biol. Chem.
PUBLISHED: 07-01-2013
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Saccharomyces cerevisiae Abz2 is a pyridoxal 5-phosphate (PLP)-dependent lyase that converts 4-amino-4-deoxychorismate (ADC) to para-aminobenzoate and pyruvate. To investigate the catalytic mechanism, we determined the 1.9 ? resolution crystal structure of Abz2 complexed with PLP, representing the first eukaryotic ADC lyase structure. Unlike Escherichia coli ADC lyase, whose dimerization is critical to the formation of the active site, the overall structure of Abz2 displays as a monomer of two domains. At the interdomain cleft, a molecule of cofactor PLP forms a Schiff base with residue Lys-251. Computational simulations defined a basic clamp to orientate the substrate ADC in a proper pose, which was validated by site-directed mutageneses combined with enzymatic activity assays. Altogether, we propose a putative catalytic mechanism of a unique class of monomeric ADC lyases led by yeast Abz2.
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Structural insights into the substrate specificity of a 6-phospho-?-glucosidase BglA-2 from Streptococcus pneumoniae TIGR4.
J. Biol. Chem.
PUBLISHED: 04-11-2013
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The 6-phospho-?-glucosidase BglA-2 (EC 3.2.1.86) from glycoside hydrolase family 1 (GH-1) catalyzes the hydrolysis of ?-1,4-linked cellobiose 6-phosphate (cellobiose-6P) to yield glucose and glucose 6-phosphate. Both reaction products are further metabolized by the energy-generating glycolytic pathway. Here, we present the first crystal structures of the apo and complex forms of BglA-2 with thiocellobiose-6P (a non-metabolizable analog of cellobiose-6P) at 2.0 and 2.4 ? resolution, respectively. Similar to other GH-1 enzymes, the overall structure of BglA-2 from Streptococcus pneumoniae adopts a typical (?/?)8 TIM-barrel, with the active site located at the center of the convex surface of the ?-barrel. Structural analyses, in combination with enzymatic data obtained from site-directed mutant proteins, suggest that three aromatic residues, Tyr(126), Tyr(303), and Trp(338), at subsite +1 of BglA-2 determine substrate specificity with respect to 1,4-linked 6-phospho-?-glucosides. Moreover, three additional residues, Ser(424), Lys(430), and Tyr(432) of BglA-2, were found to play important roles in the hydrolytic selectivity toward phosphorylated rather than non-phosphorylated compounds. Comparative structural analysis suggests that a tryptophan versus a methionine/alanine residue at subsite -1 may contribute to the catalytic and substrate selectivity with respect to structurally similar 6-phospho-?-galactosidases and 6-phospho-?-glucosidases assigned to the GH-1 family.
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Structures of yeast Apa2 reveal catalytic insights into a canonical AP?A phosphorylase of the histidine triad superfamily.
J. Mol. Biol.
PUBLISHED: 03-06-2013
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The homeostasis of intracellular diadenosine 5,5?-P(1),P(4)-tetraphosphate (Ap4A) in the yeast Saccharomyces cerevisiae is maintained by two 60% sequence-identical paralogs of Ap4A phosphorylases (Apa1 and Apa2). Enzymatic assays show that, compared to Apa1, Apa2 has a relatively higher phosphorylase activity towards Ap3A (5,5?-P(1),P(3)-tetraphosphate), Ap4A, and Ap5A (5,5?-P(1),P(5)-tetraphosphate), and Ap4A is the favorable substrate for both enzymes. To decipher the catalytic insights, we determined the crystal structures of Apa2 in the apo-, AMP-, and Ap4A-complexed forms at 2.30, 2.80, and 2.70Å resolution, respectively. Apa2 is an ?/? protein with a core domain of a twisted eight-stranded antiparallel ?-sheet flanked by several ?-helices, similar to the galactose-1-phosphate uridylyltransferase (GalT) members of the histidine triad (HIT) superfamily. However, a unique auxiliary domain enables an individual Apa2 monomer to possess an intact substrate-binding cleft, which is distinct from previously reported dimeric GalT proteins. This cleft is perfectly complementary to the favorable substrate Ap4A, the AMP and ATP moieties of which are perpendicular to each other, leaving the ?-phosphate group exposed at the sharp turn against the catalytic residue His161. Structural comparisons combined with site-directed mutagenesis and activity assays enable us to define the key residues for catalysis. Furthermore, multiple-sequence alignment reveals that Apa2 and homologs represent canonical Ap4A phosphorylases, which could be grouped as a unique branch in the GalT family.
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The N-terminal ?-sheet of peroxiredoxin 4 in the large yellow croaker Pseudosciaena crocea is involved in its biological functions.
PLoS ONE
PUBLISHED: 01-16-2013
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Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that exhibit peroxidase and peroxynitrite reductase activities involved in the reduction of reactive oxygen species. The peroxiredoxin Prx4 from the large yellow croaker Pseudosciaena crocea is a typical 2-Cys Prx with an N-terminal signal peptide. We solved the crystal structure of Prx4 at 1.90 Å and revealed an N-terminal antiparallel ?-sheet that contributes to the dimer interface. Deletion of this ?-sheet decreased the in vitro peroxidase activity to about 50% of the wild-type. In vivo assays further demonstrated that removal of this ?-sheet led to some impairment in the ability of Prx4 to negatively regulate nuclear factor-?B (NF-?B) activity and to perform its role in anti-bacterial immunity. These results provide new insights into the structure and function relationship of a peroxiredoxin from bony fish.
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A c-Myc-MicroRNA functional feedback loop affects hepatocarcinogenesis.
Hepatology
PUBLISHED: 01-12-2013
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c-Myc (Myc) plays an important role in normal liver development and tumorigenesis. We show here that Myc is pathologically activated in and essential for promoting human hepatocellular carcinoma (HCC). Myc induces HCC through a novel, microRNA (miRNA)-mediated feedback loop comprised of miR-148a-5p, miR-363-3p, and ubiquitin-specific protease 28 (USP28). Myc directly binds to conserved regions in the promoters of the two miRNAs and represses their expression. miR-148a-5p directly targets and inhibits Myc, whereas miR-363-3p destabilizes Myc by directly targeting and inhibiting USP28. Inhibition of miR-148a-5p or miR-363-3p induces hepatocellular tumorigenesis by promoting G1 to S phase progression, whereas activation of them has the opposite effects. The Myc-miRNA feedback loop is dysregulated in human HCC. Conclusion: These results define miR-148a-5p and miR-363-3p as negative regulators of Myc, thus revealing their heretofore unappreciated roles in hepatocarcinogenesis. (HEPATOLOGY 2013;57:2378-2389).
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Structures of Streptococcus pneumoniae PiaA and its complex with ferrichrome reveal insights into the substrate binding and release of high affinity iron transporters.
PLoS ONE
PUBLISHED: 01-01-2013
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Iron scarcity is one of the nutrition limitations that the Gram-positive infectious pathogens Streptococcus pneumoniae encounter in the human host. To guarantee sufficient iron supply, the ATP binding cassette (ABC) transporter Pia is employed to uptake iron chelated by hydroxamate siderophore, via the membrane-anchored substrate-binding protein PiaA. The high affinity towards ferrichrome enables PiaA to capture iron at a very low concentration in the host. We presented here the crystal structures of PiaA in both apo and ferrichrome-complexed forms at 2.7 and 2.1 Å resolution, respectively. Similar to other class III substrate binding proteins, PiaA is composed of an N-terminal and a C-terminal domain bridged by an ?-helix. At the inter-domain cleft, a molecule of ferrichrome is stabilized by a number of highly conserved residues. Upon ferrichrome binding, two highly flexible segments at the entrance of the cleft undergo significant conformational changes, indicating their contribution to the binding and/or release of ferrichrome. Superposition to the structure of Escherichia coli ABC transporter BtuF enabled us to define two conserved residues: Glu119 and Glu262, which were proposed to form salt bridges with two arginines of the permease subunits. Further structure-based sequence alignment revealed that the ferrichrome binding pattern is highly conserved in a series of PiaA homologs encoded by both Gram-positive and negative bacteria, which were predicted to be sensitive to albomycin, a sideromycin antibiotic derived from ferrichrome.
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Structural basis for the substrate specificity of a novel ?-N-acetylhexosaminidase StrH protein from Streptococcus pneumoniae R6.
J. Biol. Chem.
PUBLISHED: 10-19-2011
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The ?-N-acetylhexosaminidase (EC 3.2.1.52) from glycoside hydrolase family 20 (GH20) catalyzes the hydrolysis of the ?-N-acetylglucosamine (NAG) group from the nonreducing end of various glycoconjugates. The putative surface-exposed N-acetylhexosaminidase StrH/Spr0057 from Streptococcus pneumoniae R6 was proved to contribute to the virulence by removal of ?(1,2)-linked NAG on host defense molecules following the cleavage of sialic acid and galactose by neuraminidase and ?-galactosidase, respectively. StrH is the only reported GH20 enzyme that contains a tandem repeat of two 53% sequence-identical catalytic domains (designated as GH20-1 and GH20-2, respectively). Here, we present the 2.1 ? crystal structure of the N-terminal domain of StrH (residues Glu-175 to Lys-642) complexed with NAG. It adopts an overall structure similar to other GH20 enzymes: a (?/?)(8) TIM barrel with the active site residing at the center of the ?-barrel convex side. The kinetic investigation using 4-nitrophenyl N-acetyl-?-d-glucosaminide as the substrate demonstrated that GH20-1 had an enzymatic activity (k(cat)/K(m)) of one-fourth compared with GH20-2. The lower activity of GH20-1 could be attributed to the substitution of active site Cys-469 of GH20-1 to the counterpart Tyr-903 of GH20-2. A complex model of NAG?(1,2)Man at the active site of GH20-1 combined with activity assays of the corresponding site-directed mutants characterized two key residues Trp-443 and Tyr-482 at subsite +1 of GH20-1 (Trp-876 and Tyr-914 of GH20-2) that might determine the ?(1,2) substrate specificity. Taken together, these findings shed light on the mechanism of catalytic specificity toward the ?(1,2)-linked ?-N-acetylglucosides.
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Structural and enzymatic characterization of the streptococcal ATP/diadenosine polyphosphate and phosphodiester hydrolase Spr1479/SapH.
J. Biol. Chem.
PUBLISHED: 08-23-2011
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Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 ? and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 ?, respectively. The core structure of Spr1479 adopts a four-layer ????-sandwich fold, with Fe(3+) and Mn(2+) coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (Ap(n)A) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/Ap(n)A hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/Ap(n)A and phosphodiester hydrolase.
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Structural insights into the cofactor-assisted substrate recognition of yeast quinone oxidoreductase Zta1.
J. Struct. Biol.
PUBLISHED: 05-17-2011
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Quinone oxidoreductase (QOR EC1.6.5.5) catalyzes the reduction of quinone to hydroxyquinone using NADPH as a cofactor. Here we present the crystal structure of the ?-crystallin-like QOR Zta1 from Saccharomycescerevisiae in apo-form at 2.00 Å and complexed with NADPH at 1.59 Å resolution. Zta1 forms a homodimer, with each subunit containing a catalytic and a cofactor-binding domain. Upon NADPH binding to the interdomain cleft, the two domains shift towards each other, producing a better fit for NADPH, and tightening substrate binding. Computational simulation combined with site-directed mutagenesis and enzymatic activity analysis defined a potential quinone-binding site that determines the stringent substrate specificity. Moreover, multiple-sequence alignment and kinetics assays implied that a single-residue change from Arg in lower organisms to Gly in vertebrates possibly resulted in elevation of enzymatic activity of ?-crystallin-like QORs throughout evolution.
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Crystal structure of the 30 K protein from the silkworm Bombyx mori reveals a new member of the ?-trefoil superfamily.
J. Struct. Biol.
PUBLISHED: 04-07-2011
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The hemolymph of the fifth instar larvae of the silkworm Bombyx mori contains a group of homologous proteins with a molecular weight of approximately 30 kDa, termed B. mori low molecular weight lipoproteins (Bmlps), which account for about 5% of the total plasma proteins. These so-called "30 K proteins" have been reported to be involved in the innate immune response and transportation of lipid and/or sugar. To elucidate their molecular functions, we determined the crystal structure of a 30 K protein, Bmlp7, at 1.91Å. It has two distinct domains: an all-? N-terminal domain (NTD) and an all-? C-terminal domain (CTD) of the ?-trefoil fold. Comparative structural analysis indicates that Bmlp7 represents a new family, adding to the 14 families currently identified, of the ?-trefoil superfamily. Structural comparison and simulation suggest that the NTD has a putative lipid-binding cavity, whereas the CTD has a potential sugar-binding site. However, we were unable to detect the binding of either lipid or sugar. Therefore, further investigations are needed to characterize the molecular function of this protein.
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Structure-guided activity restoration of the silkworm glutathione transferase Omega GSTO3-3.
J. Mol. Biol.
PUBLISHED: 04-02-2011
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Glutathione transferases (GSTs) are ubiquitous detoxification enzymes that conjugate hydrophobic xenobiotics with reduced glutathione. The silkworm Bombyx mori encodes four isoforms of GST Omega (GSTO), featured with a catalytic cysteine, except that bmGSTO3-3 has an asparagine substitution of this catalytic residue. Here, we determined the 2.20-Å crystal structure of bmGSTO3-3, which shares a typical GST overall structure. However, the extended C-terminal segment that exists in all the four bmGSTOs occupies the G-site of bmGSTO3-3 and makes it unworkable, as shown by the activity assays. Upon mutation of Asn29 to Cys and truncation of the C-terminal segment, the in vitro GST activity of bmGSTO3-3 could be restored. These findings provided structural insights into the activity regulation of GSTOs.
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Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33.
J. Struct. Biol.
PUBLISHED: 03-05-2011
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The yeast Saccharomyces cerevisiae mitochondrial matrix factor Mmf1, a member in the YER057c/Yigf/Uk114 family, participates in isoleucine biosynthesis and mitochondria maintenance. Mmf1 physically interacts with another mitochondrial matrix protein Mam33, which is involved in the sorting of cytochrome b? to the intermembrane space as well as mitochondrial ribosomal protein synthesis. To elucidate the structural basis for their interaction, we determined the crystal structures of Mmf1 and Mam33 at 1.74 and 2.10 Å, respectively. Both Mmf1 and Mam33 adopt a trimeric structure: each subunit of Mmf1 displays a chorismate mutase fold with a six-stranded ?-sheet flanked by two ?-helices on one side, whereas a subunit of Mam33 consists of a twisted six-stranded ?-sheet surrounded by five ?-helices. Biochemical assays combined with structure-based computational simulation enable us to model a putative complex of Mmf1-Mam33, which consists of one Mam33 trimer and two tandem Mmf1 trimers in a head-to-tail manner. The two interfaces between the ring-like trimers are mainly composed of electrostatic interactions mediated by complementary negatively and positively charged patches. These results provided the structural insights into the putative function of Mmf1 during mitochondrial protein synthesis via Mam33, a protein binding to mitochondrial ribosomal proteins.
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Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC.
Biochem. J.
PUBLISHED: 03-02-2011
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The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two ?/?/? globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes themultiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr?? in OpuCC compared with Asp?? in OpuBC.
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Structural plasticity of the thioredoxin recognition site of yeast methionine S-sulfoxide reductase Mxr1.
J. Biol. Chem.
PUBLISHED: 02-23-2011
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The methionine S-sulfoxide reductase MsrA catalyzes the reduction of methionine sulfoxide, a ubiquitous reaction depending on the thioredoxin system. To investigate interactions between MsrA and thioredoxin (Trx), we determined the crystal structures of yeast MsrA/Mxr1 in their reduced, oxidized, and Trx2-complexed forms, at 2.03, 1.90, and 2.70 ?, respectively. Comparative structure analysis revealed significant conformational changes of the three loops, which form a plastic "cushion" to harbor the electron donor Trx2. The flexible C-terminal loop enabled Mxr1 to access the methionine sulfoxide on various protein substrates. Moreover, the plasticity of the Trx binding site on Mxr1 provides structural insights into the recognition of diverse substrates by a universal catalytic motif of Trx.
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Structural insights into the catalytic mechanism of the yeast pyridoxal 5-phosphate synthase Snz1.
Biochem. J.
PUBLISHED: 10-06-2010
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In most eubacteria, fungi, apicomplexa, plants and some metazoans, the active form of vitamin B6, PLP (pyridoxal 5-phosphate), is de novo synthesized from three substrates, R5P (ribose 5-phosphate), DHAP (dihydroxyacetone phosphate) and ammonia hydrolysed from glutamine by a complexed glutaminase. Of the three active sites of DXP (deoxyxylulose 5-phosphate)independent PLP synthase (Pdx1), the R5P isomerization site has been assigned, but the sites for DHAP isomerization and PLP formation remain unknown. In the present study, we present the crystal structures of yeast Pdx1/Snz1, in apo-, G3P (glyceraldehyde 3-phosphate)- and PLP-bound forms, at 2.3, 1.8 and 2.2 Å (1 Å=0.1 nm) respectively. Structural and biochemical analysis enabled us to assign the PLP-formation site, a G3P-binding site and a G3P-transfer site. We propose a putative catalytic mechanism for Pdx1/Snz1 in which R5P and DHAP are isomerized at two distinct sites and transferred along well-defined routes to a final destination for PLP synthesis.
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Crystal structure of the mucin-binding domain of Spr1345 from Streptococcus pneumoniae.
J. Struct. Biol.
PUBLISHED: 09-11-2010
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The surface protein Spr1345 from Streptococcus pneumoniae R6 is a 22-kDa mucin-binding protein (MucBP) involved in adherence and colonization of the human lung and respiratory tract. It is composed of a mucin-binding domain (MucBD) and a proline-rich domain (PRD) followed by an LPxTG motif, which is recognized and cleaved by sortase, resulting in a mature form of 171 residues (MF171) that is anchored to the cell wall. We found that the MucBD alone possesses comparable in vitro mucin-binding affinity to the mature form, and can be specifically enriched at the surface of human lung carcinoma A549 cells. Using single-wavelength anomalous dispersion (SAD) phasing method with the iodine signals, we solved the crystal structure of the MucBD at 2.0Å resolution, the first structure of MucBDs from pathogenic bacteria. The overall structure adopts an immunoglobulin-like fold with an elongated rod-like shape, composed of six anti-parallel ?-strands and a long loop. Structural comparison suggested that the conserved C-terminal moiety may participate in the recognition of mucins. These findings provided structural insights into host-pathogen interaction mediated by mucins, which might be useful for designing novel vaccines and antibiotic drugs against human diseases caused by pneumococci.
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Structural basis for the allosteric control of the global transcription factor NtcA by the nitrogen starvation signal 2-oxoglutarate.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-28-2010
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2-oxogluatarate (2-OG), a metabolite of the highly conserved Krebs cycle, not only plays a critical role in metabolism, but also constitutes a signaling molecule in a variety of organisms ranging from bacteria to plants and animals. In cyanobacteria, the accumulation of 2-OG constitutes the signal of nitrogen starvation and NtcA, a global transcription factor, has been proposed as a putative receptor for 2-OG. Here we present three crystal structures of NtcA from the cyanobacterium Anabaena: the apoform, and two ligand-bound forms in complex with either 2-OG or its analogue 2,2-difluoropentanedioic acid. All structures assemble as homodimers, with each subunit composed of an N-terminal effector-binding domain and a C-terminal DNA-binding domain connected by a long helix (C-helix). The 2-OG binds to the effector-binding domain at a pocket similar to that used by cAMP in catabolite activator protein, but with a different pattern. Comparative structural analysis reveals a putative signal transmission route upon 2-OG binding. A tighter coiled-coil conformation of the two C-helices induced by 2-OG is crucial to maintain the proper distance between the two F-helices for DNA recognition. Whereas catabolite activator protein adopts a transition from off-to-on state upon cAMP binding, our structural analysis explains well why NtcA can bind to DNA even in its apoform, and how 2-OG just enhances the DNA-binding activity of NtcA. These findings provided the structural insights into the function of a global transcription factor regulated by 2-OG, a metabolite standing at a crossroad between carbon and nitrogen metabolisms.
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Crystal structure of the cyanobacterial signal transduction protein PII in complex with PipX.
J. Mol. Biol.
PUBLISHED: 06-25-2010
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P(II) proteins are highly conserved signal transducers in bacteria, archaea, and plants. They have a large flexible loop (T-loop) that adopts different conformations after covalent modification or binding to different effectors to regulate the functions of diverse protein partners. The P(II) partner PipX (P(II)interaction protein X), first identified from Synechococcus sp. PCC 7942, exists uniquely in cyanobacteria. PipX also interacts with the cyanobacterial global nitrogen regulator NtcA. The mutually exclusive binding of P(II) and NtcA by PipX in a 2-oxoglutarate (2-OG)-dependent manner enables P(II) to indirectly regulate the transcriptional activity of NtcA. However, the structural basis for these exclusive interactions remains unknown. We solved the crystal structure of the P(II)-PipX complex from the filamentous cyanobacterium Anabaena sp. PCC 7120 at 1.90 Å resolution. A homotrimeric P(II) captures three subunits of PipX through the T-loops. Similar to P(II) from Synechococcus, the core structure consists of an antiparallel ?-sheet with four ?-strands and two ?-helices at the lateral surface. PipX adopts a novel structure composed of five twisted antiparallel ?-strands and two ?-helices, which is reminiscent of the P(II) structure. The T-loop of each P(II) subunit extends from the core structure as an antenna that is stabilized at the cleft between two PipX monomers via hydrogen bonds. In addition, the interfaces between the ?-sheets of PipX and P(II) core structures partially contribute to complex formation. Comparative structural analysis indicated that PipX and 2-OG share a common binding site that overlaps with the 14 signature residues of cyanobacterial P(II) proteins. Our structure of PipX and the recently solved NtcA structure enabled us to propose a putative model for the NtcA-PipX complex. Taken together, these findings provide structural insights into how P(II) regulates the transcriptional activity of NtcA via PipX upon accumulation of the metabolite 2-OG.
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Human and viral nucleoside/nucleotide kinases involved in antiviral drug activation: structural and catalytic properties.
Antiviral Res.
PUBLISHED: 01-31-2010
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Antiviral nucleoside and nucleotide analogs, essential for the treatment of viral infections in the absence of efficient vaccines, are prodrug forms of the active compounds that target the viral DNA polymerase or reverse transcriptase. The activation process requires several successive phosphorylation steps catalyzed by different kinases, which are present in the host cell or encoded by some of the viruses. These activation reactions often are rate-limiting steps and are thus open to improvement. We review here the structural and enzymatic properties of the enzymes that carry out the activation of analogs used in therapy against human immunodeficiency virus and against DNA viruses such as hepatitis B, herpes and poxviruses. Four major classes of drugs are considered: thymidine analogs, non-natural L-nucleosides, acyclic nucleoside analogs and acyclic nucleoside phosphonate analogs. Their efficiency as drugs depends both on the low specificity of the viral polymerase that allows their incorporation into DNA, but also on the ability of human/viral kinases to provide the activated triphosphate active forms at a high concentration at the right place. Two distinct modes of action are considered, depending on the origin of the kinase (human or viral). If the human kinases are house-keeping enzymes that belong to the metabolic salvage pathway, herpes and poxviruses encode for related enzymes. The structures, substrate specificities and catalytic properties of each of these kinases are discussed in relation to drug activation.
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Structural and biochemical characterization of yeast monothiol glutaredoxin Grx6.
J. Mol. Biol.
PUBLISHED: 01-06-2010
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Glutaredoxins (Grxs) are a ubiquitous family of proteins that reduce disulfide bonds in substrate proteins using electrons from reduced glutathione (GSH). The yeast Saccharomyces cerevisiae Grx6 is a monothiol Grx that is localized in the endoplasmic reticulum and Golgi compartments. Grx6 consists of three segments, a putative signal peptide (M1-I36), an N-terminal domain (K37-T110), and a C-terminal Grx domain (K111-N231, designated Grx6C). Compared to the classic dithiol glutaredoxin Grx1, Grx6 has a lower glutathione disulfide reductase activity but a higher glutathione S-transferase activity. In addition, similar to human Grx2, Grx6 binds GSH via an iron-sulfur cluster in vitro. The N-terminal domain is essential for noncovalent dimerization, but not required for either of the above activities. The crystal structure of Grx6C at 1.5 A resolution revealed a novel two-strand antiparallel beta-sheet opposite the GSH binding groove. This extra beta-sheet might also exist in yeast Grx7 and in a group of putative Grxs in lower organisms, suggesting that Grx6 might represent the first member of a novel Grx subfamily.
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Structural insights into the substrate tunnel of Saccharomyces cerevisiae carbonic anhydrase Nce103.
BMC Struct. Biol.
PUBLISHED: 10-24-2009
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The carbonic anhydrases (CAs) are involved in inorganic carbon utilization. They have been classified into six evolutionary and structural families: alpha-, beta-, gamma-, delta-, epsilon-, zeta- CAs, with beta-CAs present in higher plants, algae and prokaryotes. The yeast Saccharomyces cerevisiae encodes a single copy of beta-CA Nce103/YNL036W.
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Nucleoside diphosphate kinase and the activation of antiviral phosphonate analogs of nucleotides: binding mode and phosphorylation of tenofovir derivatives.
Nucleosides Nucleotides Nucleic Acids
PUBLISHED: 08-28-2009
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Tenofovir is an acyclic phosphonate analog of deoxyadenylate used in AIDS and hepatitis B therapy. We find that tenofovir diphosphate, its active form, can be produced by human nucleoside diphosphate kinase (NDPK), but with low efficiency, and that creatine kinase is significantly more active. The 1.65 A x-ray structure of NDPK in complex with tenofovir mono- and diphosphate shows that the analogs bind at the same site as natural nucleotides, but in a different conformation, and make only a subset of the Van der Waals and polar interactions made by natural substrates, consistent with their comparatively low affinity for the enzyme.
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The ternary structure of the double-headed arrowhead protease inhibitor API-A complexed with two trypsins reveals a novel reactive site conformation.
J. Biol. Chem.
PUBLISHED: 07-28-2009
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The double-headed arrowhead protease inhibitors API-A and -B from the tubers of Sagittaria sagittifolia (Linn) feature two distinct reactive sites, unlike other members of their family. Although the two inhibitors have been extensively characterized, the identities of the two P1 residues in both API-A and -B remain controversial. The crystal structure of a ternary complex at 2.48 A resolution revealed that the two trypsins bind on opposite sides of API-A and are 34 A apart. The overall fold of API-A belongs to the beta-trefoil fold and resembles that of the soybean Kunitz-type trypsin inhibitors. The two P1 residues were unambiguously assigned as Leu(87) and Lys(145), and their identities were further confirmed by site-directed mutagenesis. Reactive site 1, composed of residues P5 Met(83) to P5 Ala(92), adopts a novel conformation with the Leu(87) completely embedded in the S1 pocket even though it is an unfavorable P1 residue for trypsin. Reactive site 2, consisting of residues P5 Cys(141) to P5 Glu(150), binds trypsin in the classic mode by employing a two-disulfide-bonded loop. Analysis of the two binding interfaces sheds light on atomic details of the inhibitor specificity and also promises potential improvements in enzyme activity by engineering of the reactive sites.
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Structure of the choline-binding domain of Spr1274 in Streptococcus pneumoniae.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 05-27-2009
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Spr1274 is a putative choline-binding protein that is bound to the cell wall of Streptococcus pneumoniae through noncovalent interactions with the choline moieties of teichoic and lipoteichoic acids. Its function is still unknown. The crystal structure of the choline-binding domain of Spr1274 (residues 44-129) was solved at 2.38 A resolution with three molecules in the asymmetric unit. It may provide a structural basis for functional analysis of choline-binding proteins.
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Structures of yeast glutathione-S-transferase Gtt2 reveal a new catalytic type of GST family.
EMBO Rep.
PUBLISHED: 05-20-2009
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Glutathione-S-transferases (GSTs) are ubiquitous detoxification enzymes that catalyse the conjugation of electrophilic substrates to glutathione. Here, we present the crystal structures of Gtt2, a GST of Saccharomyces cerevisiae, in apo and two ligand-bound forms, at 2.23 A, 2.20 A and 2.10 A, respectively. Although Gtt2 has the overall structure of a GST, the absence of the classic catalytic essential residues--tyrosine, serine and cysteine--distinguishes it from all other cytosolic GSTs of known structure. Site-directed mutagenesis in combination with activity assays showed that instead of the classic catalytic residues, a water molecule stabilized by Ser129 and His123 acts as the deprotonator of the glutathione sulphur atom. Furthermore, only glycine and alanine are allowed at the amino-terminus of helix-alpha1 because of stereo-hindrance. Taken together, these results show that yeast Gtt2 is a novel atypical type of cytosolic GST.
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Crystal structure of Saccharomyces cerevisiae cytoplasmic thioredoxin reductase Trr1 reveals the structural basis for species-specific recognition of thioredoxin.
Biochim. Biophys. Acta
PUBLISHED: 03-14-2009
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Thioredoxin reductase (TrxR) is a member of the pyridine nucleotide-disulfide oxidoreductase family of the flavoenzymes. It can use a dithiol-disulfide active-site to transfer reducing equivalents from NADPH to thioredoxin (Trx), via the cofactor FAD. In Saccharomyces cerevisiae, the cytoplasmic thioredoxin reductase Trr1 plays an important role in multiple cellular events under the control of transcription factor Yap1 and/or Rho5. Here we present the crystal structure of Trr1 at the resolution of 2.8 A, the first fungal TrxR structure. Structural analysis shows it shares a very similar overall structure to Escherichia coli TrxR. However, fine comparisons indicate some distinct differences at the Trx recognition sites. These differences might be responsible to the species-specific recognition of Trx, which has been demonstrated by previous biochemical assays.
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Structure of the thioredoxin-fold domain of human phosducin-like protein 2.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 01-31-2009
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Human phosducin-like protein 2 (hPDCL2) has been identified as belonging to subgroup II of the phosducin (Pdc) family. The members of this family share an N-terminal helix domain and a C-terminal thioredoxin-fold (Trx-fold) domain. The X-ray crystal structure of the Trx-fold domain of hPDCL2 was solved at 2.70 A resolution and resembled the Trx-fold domain of rat phosducin. Comparative structural analysis revealed the structural basis of their putative functional divergence.
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Structural and kinetic analysis of Saccharomyces cerevisiae thioredoxin Trx1: implications for the catalytic mechanism of GSSG reduced by the thioredoxin system.
Biochim. Biophys. Acta
PUBLISHED: 01-24-2009
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Thioredoxin (Trx) and glutathione/glutaredoxin (GSH/Grx) systems play the dominant role in cellular redox homeostasis. Recently the Trx system has been shown to be responsible to control the balance of GSH/GSSG once the glutathione reductase system is not available. To decipher the structural basis of electron transfer from the Trx system to GSSG, we solved the crystal structures of oxidized Trx1 and glutathionylated Trx1Cys33Ser mutant at 1.76 and 1.80 A, respectively. Comparative structural analysis revealed a key residue Met35 involved in the Trx-GSSG recognition. Subsequent mutagenesis and kinetic studies proved that Met35Arg mutation could alter the apparent K(m) and V(max) values of the reaction. These findings gave us the structural insights into GSSG reduction catalyzed by the Trx system.
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Purification, crystallization and preliminary X-ray diffraction analysis of glutathionylated Trx1 C33S mutant from yeast.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
PUBLISHED: 01-21-2009
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Thioredoxins (Trxs) are a family of small redox-active proteins that are found in all living organisms. In Saccharomyces cerevisiae, two cytosolic Trxs (Trx1 and Trx2) and one mitochondrial Trx (Trx3) have previously been identified. In this work, cytosolic Trx1 containing a C33S mutant was overexpressed, purified, glutathionylated and crystallized using the hanging-drop vapour-diffusion method. A set of X-ray diffraction data was collected to 1.80 A resolution. The crystal belonged to space group P1, with unit-cell parameters a = 38.53, b = 38.81, c = 41.70 A, alpha = 72.91, beta = 87.51, gamma = 60.58 degrees.
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Structural and mechanistic analyses of yeast mitochondrial thioredoxin Trx3 reveal putative function of its additional cysteine residues.
Biochim. Biophys. Acta
PUBLISHED: 01-03-2009
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The yeast Saccharomyces cerevisiae Trx3 is a key member of the thioredoxin system to control the cellular redox homeostasis in mitochondria. We solved the crystal structures of yeast Trx3 in oxidized and reduced forms at 1.80 and 2.10 A, respectively. Besides the active site, the additional cysteine residue Cys69 also undergoes a significant redox-correlated conformational change. Comparative structural analyses in combination with activity assays revealed that residue Cys69 could be S-nitrosylated in vitro. S-nitrosylation of Cys69 will decrease the activity of Trx3 by 20%, which is comparable to the effect of the Cys69Ser mutation. Taken together, these findings provided us some new insights into the putative function of the additional cysteine residues of Trx3.
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Structure of yeast sulfhydryl oxidase erv1 reveals electron transfer of the disulfide relay system in the mitochondrial intermembrane space.
J. Biol. Chem.
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The disulfide relay system in the mitochondrial intermembrane space drives the import of proteins with twin CX(9)C or twin CX(3)C motifs by an oxidative folding mechanism. This process requires disulfide bond transfer from oxidized Mia40 to a substrate protein. Reduced Mia40 is reoxidized/regenerated by the FAD-linked sulfhydryl oxidase Erv1 (EC 1.8.3.2). Full-length Erv1 consists of a flexible N-terminal shuttle domain (NTD) and a conserved C-terminal core domain (CTD). Here, we present crystal structures at 2.0 ? resolution of the CTD and at 3.0 ? resolution of a C30S/C133S double mutant of full-length Erv1 (Erv1FL). Similar to previous homologous structures, the CTD exists as a homodimer, with each subunit consisting of a conserved four-helix bundle that accommodates the isoalloxazine ring of FAD and an additional single-turn helix. The structure of Erv1FL enabled us to identify, for the first time, the three-dimensional structure of the Erv1NTD, which is an amphipathic helix flanked by two flexible loops. This structure also represents an intermediate state of electron transfer from the NTD to the CTD of another subunit. Comparative structural analysis revealed that the four-helix bundle of the CTD forms a wide platform for the electron donor NTD. Moreover, computational simulation combined with multiple-sequence alignment suggested that the amphipathic helix close to the shuttle redox enter is critical for the recognition of Mia40, the upstream electron donor. These findings provide structural insights into electron transfer from Mia40 via the shuttle domain of one subunit of Erv1 to the CTD of another Erv1 subunit.
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Structural insights into the substrate specificity of Streptococcus pneumoniae ?(1,3)-galactosidase BgaC.
J. Biol. Chem.
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The surface-exposed ?-galactosidase BgaC from Streptococcus pneumoniae was reported to be a virulence factor because of its specific hydrolysis activity toward the ?(1,3)-linked galactose and N-acetylglucosamine (Gal?(1,3)NAG) moiety of oligosaccharides on the host molecules. Here we report the crystal structure of BgaC at 1.8 ? and its complex with galactose at 1.95 ?. At pH 5.5-8.0, BgaC exists as a stable homodimer, each subunit of which consists of three distinct domains: a catalytic domain of a classic (?/?)(8) TIM barrel, followed by two all-? domains (ABDs) of unknown function. The side walls of the TIM ?-barrel and a loop extended from the first ABD constitute the active site. Superposition of the galactose-complexed structure to the apo-form revealed significant conformational changes of residues Trp-243 and Tyr-455. Simulation of a putative substrate entrance tunnel and modeling of a complex structure with Gal?(1,3)NAG enabled us to assign three key residues to the specific catalysis. Site-directed mutagenesis in combination with activity assays further proved that residues Trp-240 and Tyr-455 contribute to stabilizing the N-acetylglucosamine moiety, whereas Trp-243 is critical for fixing the galactose ring. Moreover, we propose that BgaC and other galactosidases in the GH-35 family share a common domain organization and a conserved substrate-determinant aromatic residue protruding from the second domain.
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Structural snapshots of yeast alkyl hydroperoxide reductase Ahp1 peroxiredoxin reveal a novel two-cysteine mechanism of electron transfer to eliminate reactive oxygen species.
J. Biol. Chem.
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Peroxiredoxins (Prxs) are thiol-specific antioxidant proteins that protect cells against reactive oxygen species and are involved in cellular signaling pathways. Alkyl hydroperoxide reductase Ahp1 belongs to the Prx5 subfamily and is a two-cysteine (2-Cys) Prx that forms an intermolecular disulfide bond. Enzymatic assays and bioinformatics enabled us to re-assign the peroxidatic cysteine (C(P)) to Cys-62 and the resolving cysteine (C(R)) to Cys-31 but not the previously reported Cys-120. Thus Ahp1 represents the first 2-Cys Prx with a peroxidatic cysteine after the resolving cysteine in the primary sequence. We also found the positive cooperativity of the substrate t-butyl hydroperoxide binding to Ahp1 homodimer at a Hill coefficient of ?2, which enabled Ahp1 to eliminate hydroperoxide at much higher efficiency. To gain the structural insights into the catalytic cycle of Ahp1, we determined the crystal structures of Ahp1 in the oxidized, reduced, and Trx2-complexed forms at 2.40, 2.91, and 2.10 ? resolution, respectively. Structural superposition of the oxidized to the reduced form revealed significant conformational changes at the segments containing C(P) and C(R). An intermolecular C(P)-C(R) disulfide bond crossing the A-type dimer interface distinguishes Ahp1 from other typical 2-Cys Prxs. The structure of the Ahp1-Trx2 complex showed for the first time how the electron transfers from thioredoxin to a peroxidase with a thioredoxin-like fold. In addition, site-directed mutagenesis in combination with enzymatic assays suggested that the peroxidase activity of Ahp1 would be altered upon the urmylation (covalently conjugated to ubiquitin-related modifier Urm1) of Lys-32.
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N-Terminal domain of Bombyx mori fibroin mediates the assembly of silk in response to pH decrease.
J. Mol. Biol.
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Fibroins serve as the major building blocks of silk fiber. As the major component of fibroin, the fibroin heavy chain is a considerably large protein comprising N-terminal and C-terminal hydrophilic domains and 12 highly repetitive Gly-Ala-rich regions flanked by internal hydrophilic blocks. Here, we show the crystal structure of the fibroin N-terminal domain (FibNT) at pH 4.7, revealing a remarkable double-layered anti-parallel ?-sheet with each layer comprising two FibNT molecules entangled together. We also show that FibNT undergoes a pH-responsive conformational transition from random coil to ?-sheets at around pH 6.0. Dynamic light scattering demonstrates that FibNT tends to oligomerize as pH decreases to 6.0, and electron microscopy reveals micelle-like oligomers. Our results are consistent with the micelle assembly model of silk fibroin and, more importantly, show that the N-terminal domain in itself has the capacity to form micelle-like structures in response to pH decrease. Structural and mutagenesis analyses further reveal the important role of conserved acidic residues clustered in FibNT, such as Glu56 and Asp100, in preventing premature ?-sheet formation at neutral pH. Collectively, we suggest that FibNT functions as a pH-responsive self-assembly module that could prevent premature ?-sheet formation at neutral pH yet could initiate fibroin assembly as pH decreases along the lumen of the posterior silk gland to the anterior silk gland.
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