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1.
Eek P Järving R Järving I Gilbert NC Newcomer ME Samel N 《The Journal of biological chemistry》2012,287(26):22377-22386
Lipoxygenases (LOXs) are a key part of several signaling pathways that lead to inflammation and cancer. Yet, the mechanisms of substrate binding and allosteric regulation by the various LOX isoforms remain speculative. Here we report the 2.47-Å resolution crystal structure of the arachidonate 11R-LOX from Gersemia fruticosa, which sheds new light on the mechanism of LOX catalysis. Our crystallographic and mutational studies suggest that the aliphatic tail of the fatty acid is bound in a hydrophobic pocket with two potential entrances. We speculate that LOXs share a common T-shaped substrate channel architecture that gives rise to the varying positional specificities. A general allosteric mechanism is proposed for transmitting the activity-inducing effect of calcium binding from the membrane-targeting PLAT (polycystin-1/lipoxygenase/α-toxin) domain to the active site via a conserved π-cation bridge. 相似文献
2.
Jung-Ung An Seung-Hye Hong Deok-Kun Oh 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2018,1863(8):823-833
Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific?oxygenated signaling compounds. 相似文献
3.
Almerinda Di Venere Eleonora Nicolai Igor Ivanov Enrico Dainese Susan Adel B.C. Angelucci Hartmut Kuhn Mauro Maccarrone Giampiero Mei 《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2014,1841(1):1-10
Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors. 相似文献
4.
Julianne Beam Amy Botta Jiayu Ye Hesham Soliman Brieanne J. Matier Mary Forrest Kathleen M. MacLeod Sanjoy Ghosh 《The Journal of biological chemistry》2015,290(38):23371-23384
Controversy exists on the benefits versus harms of n-6 polyunsaturated fatty acids (n-6 PUFA). Although n-6 PUFA demonstrates anti-atherosclerotic properties, survival following cardiac remodeling may be compromised. We hypothesized that n-6 PUFA like linoleic acid (LA) or other downstream PUFAs like γ-linolenic acid or arachidonic acid alter the transforming growth factor-β (TGFβ)-collagen axis in the heart. Excess dietary LA increased the collagen I/III ratio in the mouse myocardium, leading to cardiac “stiffening” characterized by impaired transmitral flow indicative of early diastolic dysfunction within 5 weeks. In vitro, LA under TGFβ1 stimulation increased collagen I and lysyl oxidase (LOX), the enzyme that cross-links soluble collagen resulting in deposited collagen. Overexpression of fatty acid desaturase 2 (fads2), which metabolizes LA to downstream PUFAs, reduced collagen deposits, LOX maturation, and activity with LA, whereas overexpressing fads1, unrelated to LA desaturation, did not. Furthermore, fads2 knockdown by RNAi elevated LOX activity and collagen deposits in fibroblasts with LA but not oleic acid, implying a buildup of LA for aggravating such pro-fibrotic effects. As direct incubation with γ-linolenic acid or arachidonic acid also attenuated collagen deposits and LOX activity, we concluded that LA itself, independent of other downstream PUFAs, promotes the pro-fibrotic effects of n-6 PUFA. Overall, these results attempt to reconcile opposing views of n-6 PUFA on the cardiovascular system and present evidence supporting a cardiac muscle-specific effect of n-6 PUFAs. Therefore, aggravation of the collagen I/III ratio and cardiac stiffening by excess n-6 PUFA represent a novel pathway of cardiac lipotoxicity caused by high n-6 PUFA diets. 相似文献
5.
Anselm E. Oberholzer Philipp Schneider Christian Siebold Ulrich Baumann Bernhard Erni 《The Journal of biological chemistry》2009,284(48):33169-33176
The bacterial phosphoenolpyruvate (PEP) sugar phosphotransferase system mediates sugar uptake and controls the carbon metabolism in response to carbohydrate availability. Enzyme I (EI), the first component of the phosphotransferase system, consists of an N-terminal protein binding domain (EIN) and a C-terminal PEP binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here we report the 2.4 Å crystal structure of the homodimeric EI from Staphylococcus aureus. EIN consists of the helical hairpin HPr binding subdomain and the phosphorylatable βα phospho-histidine (P-His) domain. EIC folds into an (βα)8 barrel. The dimer interface of EIC buries 1833 Å2 of accessible surface per monomer and contains two Ca2+ binding sites per dimer. The structures of the S. aureus and Escherichia coli EI domains (Teplyakov, A., Lim, K., Zhu, P. P., Kapadia, G., Chen, C. C., Schwartz, J., Howard, A., Reddy, P. T., Peterkofsky, A., and Herzberg, O. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 16218–16223) are very similar. The orientation of the domains relative to each other, however, is different. In the present structure the P-His domain is docked to the HPr binding domain in an orientation appropriate for in-line transfer of the phosphate to the active site histidine of the acceptor HPr. In the E. coli structure the phospho-His of the P-His domain projects into the PEP binding site of EIC. In the S. aureus structure the crystallographic temperature factors are lower for the HPr binding domain in contact with the P-His domain and higher for EIC. In the E. coli structure it is the reverse. 相似文献
6.
Zhou K Gao Y Hoy JA Mann FM Honzatko RB Peters RJ 《The Journal of biological chemistry》2012,287(9):6840-6850
Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg2+ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities. 相似文献
7.
Effects of exogenous auxins on expression of lipoxygenases in cultured soybean embryos 总被引:4,自引:0,他引:4
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The expression of lipoxygenases (LOXs) is known to be developmentally regulated in soybeans (Glycine max. [L.] Merr.). Hormones have been firmly established as being involved in the growth and developmental processes of a number of plant species. Correlation between the expression of LOXs and the development and germination of soybean embryos suggests that plant hormones may affect the expression of LOXs. The present studies were conducted to investigate the effects of exogenous auxins on the expression of LOX isozymes and LOX activities in cultured cotyledon tissues of immature soybean seeds. The results revealed that at least one of the more acidic nonembryo LOX isozymes was induced by either α-naphthaleneacetic acid or indoleacetic acid but not by 2,4-dichlorophenoxyacetic acid after 4 days' exposure. Levels of LOX-1, -2, and -3 proteins and activities were significantly decreased by 2,4-dichlorophenoxyacetic acid 10 days after explanting. S1 analysis showed that embryo LOX messenger RNAs were detectable in the tissues treated with each of the auxins. The reduced levels of the embryo LOX proteins may, therefore, be regulated at the levels of translation, posttranslational modification, or degradation. The more acidic isozymes induced by α-naphthaleneacetic acid showed enzymatic activity and shared the same molecular mass and isoelectric point values as the germination-associated LOX isozymes found in hypocotyls and radicles, suggesting that those LOXs are involved in germination competency of soybean embryos. 相似文献
8.
Christiane Kummer Brian G. Petrich David M. Rose Mark H. Ginsberg 《The Journal of biological chemistry》2010,285(13):9462-9469
Extracellular antagonists of α4 integrin are an effective therapy for several autoimmune and inflammatory diseases; however, these agents that directly block ligand binding may exhibit mechanism-based toxicities. Inhibition of α4 integrin signaling by mutations of α4 that block paxillin binding inhibits inflammation while limiting mechanism-based toxicities. Here, we test a pharmacological approach by identifying small molecules that inhibit the α4 integrin-paxillin interaction. By screening a large (∼40,000-compound) chemical library, we identified a noncytotoxic inhibitor of this interaction that impaired integrin α4-mediated but not αLβ2-mediated Jurkat T cell migration. The identified compound had no effect on α4-mediated migration in cells bearing the α4(Y991A) mutation that disrupts the α4-paxillin interaction, establishing the specificity of its action. Administration of this compound to mice led to impaired recruitment of mononuclear leukocytes to a site of inflammation in vivo, whereas an isomer that does not inhibit the α4-paxillin interaction had no effect on α4-mediated cell migration, cell spreading, or recruitment of leukocytes to an inflammatory site. Thus, a small molecule inhibitor that interferes with α4 integrin signaling reduces α4-mediated T cell migration in vivo, thus providing proof of principle for inhibition of α4 integrin signaling as a target for the pharmacological reduction of inflammation. 相似文献
9.
Mast N Annalora AJ Lodowski DT Palczewski K Stout CD Pikuleva IA 《The Journal of biological chemistry》2011,286(7):5607-5613
Mitochondrial cytochrome P450 11A1 (CYP11A1 or P450 11A1) is the only known enzyme that cleaves the side chain of cholesterol, yielding pregnenolone, the precursor of all steroid hormones. Pregnenolone is formed via three sequential monooxygenation reactions that involve the progressive production of 22R-hydroxycholesterol (22HC) and 20α,22R-dihydroxycholesterol, followed by the cleavage of the C20-C22 bond. Herein, we present the 2.5-Å crystal structure of CYP11A1 in complex with the first reaction intermediate, 22HC. The active site cavity in CYP11A1 represents a long curved tube that extends from the protein surface to the heme group, the site of catalysis. 22HC occupies two-thirds of the cavity with the 22R-hydroxyl group nearest the heme, 2.56 Å from the iron. The space at the entrance to the active site is not taken up by 22HC but filled with ordered water molecules. The network formed by these water molecules allows the “soft” recognition of the 22HC 3β-hydroxyl. Such a mode of 22HC binding suggests shuttling of the sterol intermediates between the active site entrance and the heme group during the three-step reaction. Translational freedom of 22HC and torsional motion of its aliphatic tail are supported by solution studies. The CYP11A1–22HC co-complex also provides insight into the structural basis of the strict substrate specificity and high catalytic efficiency of the enzyme and highlights conserved structural motifs involved in redox partner interactions by mitochondrial P450s. 相似文献
10.
11.
Tijs van Wieringen Sebastian Kalamajski ?sa Lidén Dominique Bihan Bengt Guss Dick Heineg?rd Richard W. Farndale Kristofer Rubin 《The Journal of biological chemistry》2010,285(46):35803-35813
Collagen fibers expose distinct domains allowing for specific interactions with other extracellular matrix proteins and cells. To investigate putative collagen domains that govern integrin αVβ3-mediated cellular interactions with native collagen fibers we took advantage of the streptococcal protein CNE that bound native fibrillar collagens. CNE specifically inhibited αVβ3-dependent cell-mediated collagen gel contraction, PDGF BB-induced and αVβ3-mediated adhesion of cells, and binding of fibronectin to native collagen. Using a Toolkit composed of overlapping, 27-residue triple helical segments of collagen type II, two CNE-binding sites present in peptides II-1 and II-44 were identified. These peptides lack the major binding site for collagen-binding β1 integrins, defined by the peptide GFOGER. Peptide II-44 corresponds to a region of collagen known to bind collagenases, discoidin domain receptor 2, SPARC (osteonectin), and fibronectin. In addition to binding fibronectin, peptide II-44 but not II-1 inhibited αVβ3-mediated collagen gel contraction and, when immobilized on plastic, supported adhesion of cells. Reduction of fibronectin expression by siRNA reduced PDGF BB-induced αVβ3-mediated contraction. Reconstitution of collagen types I and II gels in the presence of CNE reduced collagen fibril diameters and fibril melting temperatures. Our data indicate that contraction proceeded through an indirect mechanism involving binding of cell-produced fibronectin to the collagen fibers. Furthermore, our data show that cell-mediated collagen gel contraction does not directly depend on the process of fibril formation. 相似文献
12.
Xianlong Gao Deepti Chaturvedi Tarun B. Patel 《The Journal of biological chemistry》2010,285(10):6970-6979
Previously we showed that the inactive form of p90 ribosomal S6 kinase 1 (RSK1) interacts with the regulatory subunit, PKARIα, of protein kinase A (PKA), whereas the active RSK1 interacts with the catalytic subunit (PKAc) of PKA. Herein, we demonstrate that the N-terminal kinase domain (NTK) of RSK1 is necessary for interactions with PKARIα. Substitution of the activation loop phosphorylation site (Ser-221) in the NTK with the negatively charged Asp residue abrogated the association between RSK1 and PKARIα. This explains the lack of an interaction between active RSK1 and PKARIα. Full-length RSK1 bound to PKARIα with an affinity of 0.8 nm. The NTK domain of RSK1 competed with PKAc for binding to the pseudosubstrate region (amino acids 93–99) of PKARIα. Overexpressed RSK1 dissociated PKAc from PKARIα, increasing PKAc activity, whereas silencing of RSK1 increased PKAc/PKARIα interactions and decreased PKAc activity. Unlike PKAc, which requires Arg-95 and -96 in the pseudosubstrate region of PKARIα for their interactions, RSK1/PKARIα association requires all four Arg residues (Arg-93–96) in the pseudosubstrate site of PKARIα. A peptide (Wt-PS) corresponding to residues 91–99 of PKARIα competed for binding of RSK1 with PKARIα both in vitro and in intact cells. Furthermore, peptide Wt-PS (but not control peptide Mut-PS), by dissociating RSK1 from PKARIα, activated RSK1 in the absence of any growth factors and protected cells from apoptosis. Thus, by competing for binding to the pseudosubstrate region of PKARIα, RSK1 regulates PKAc activity in a cAMP-independent manner, and PKARIα by associating with RSK1 regulates its activation and its biological functions. 相似文献
13.
Wheatley RW Zheng RB Richards MR Lowary TL Ng KK 《The Journal of biological chemistry》2012,287(33):28132-28143
Biosynthesis of the mycobacterial cell wall relies on the activities of many enzymes, including several glycosyltransferases (GTs). The polymerizing galactofuranosyltransferase GlfT2 (Rv3808c) synthesizes the bulk of the galactan portion of the mycolyl-arabinogalactan complex, which is the largest component of the mycobacterial cell wall. We used x-ray crystallography to determine the 2.45-Å resolution crystal structure of GlfT2, revealing an unprecedented multidomain structure in which an N-terminal β-barrel domain and two primarily α-helical C-terminal domains flank a central GT-A domain. The kidney-shaped protomers assemble into a C4-symmetric homotetramer with an open central core and a surface containing exposed hydrophobic and positively charged residues likely involved with membrane binding. The structure of a 3.1-Å resolution complex of GlfT2 with UDP reveals a distinctive mode of nucleotide recognition. In addition, models for the binding of UDP-galactofuranose and acceptor substrates in combination with site-directed mutagenesis and kinetic studies suggest a mechanism that explains the unique ability of GlfT2 to generate alternating β-(1→5) and β-(1→6) glycosidic linkages using a single active site. The topology imposed by docking a tetrameric assembly onto a membrane bilayer also provides novel insights into aspects of processivity and chain length regulation in this and possibly other polymerizing GTs. 相似文献
14.
?yvind Halskau Jr. Ming Ying Anne Baumann Rune Kleppe David Rodriguez-Larrea Bj?rg Alm?s Jan Haavik Aurora Martinez 《The Journal of biological chemistry》2009,284(47):32758-32769
Tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of catecholamines, is activated by phosphorylation-dependent binding to 14-3-3 proteins. The N-terminal domain of TH is also involved in interaction with lipid membranes. We investigated the binding of the N-terminal domain to its different partners, both in the unphosphorylated (TH-(1–43)) and Ser19-phosphorylated (THp-(1–43)) states by surface plasmon resonance. THp-(1–43) showed high affinity for 14-3-3 proteins (Kd ∼ 0.5 μm for 14-3-3γ and -ζ and 7 μm for 14-3-3η). The domains also bind to negatively charged membranes with intermediate affinity (concentration at half-maximal binding S0.5 = 25–58 μm (TH-(1–43)) and S0.5 = 135–475 μm (THp-(1–43)), depending on phospholipid composition) and concomitant formation of helical structure. 14-3-3γ showed a preferential binding to membranes, compared with 14-3-3ζ, both in chromaffin granules and with liposomes at neutral pH. The affinity of 14-3-3γ for negatively charged membranes (S0.5 = 1–9 μm) is much higher than the affinity of TH for the same membranes, compatible with the formation of a ternary complex between Ser19-phosphorylated TH, 14-3-3γ, and membranes. Our results shed light on interaction mechanisms that might be relevant for the modulation of the distribution of TH in the cytoplasm and membrane fractions and regulation of l-DOPA and dopamine synthesis. 相似文献
15.
Young-Hyun Han Marie-Line Garron Hye-Yeon Kim Wan-Seok Kim Zhenqing Zhang Kyeong-Seok Ryu David Shaya Zhongping Xiao Chaejoon Cheong Yeong Shik Kim Robert J. Linhardt Young Ho Jeon Miroslaw Cygler 《The Journal of biological chemistry》2009,284(49):34019-34027
Heparin lyase I (heparinase I) specifically depolymerizes heparin, cleaving the glycosidic linkage next to iduronic acid. Here, we show the crystal structures of heparinase I from Bacteroides thetaiotaomicron at various stages of the reaction with heparin oligosaccharides before and just after cleavage and product disaccharide. The heparinase I structure is comprised of a β-jellyroll domain harboring a long and deep substrate binding groove and an unusual thumb-resembling extension. This thumb, decorated with many basic residues, is of particular importance in activity especially on short heparin oligosaccharides. Unexpected structural similarity of the active site to that of heparinase II with an (α/α)6 fold is observed. Mutational studies and kinetic analysis of this enzyme provide insights into the catalytic mechanism, the substrate recognition, and processivity. 相似文献
16.
Yu-Min Lin Shih-Jung Wu Ting-Wei Chang Chiu-Feng Wang Ching-Shu Suen Ming-Jing Hwang Margaret Dah-Tsyr Chang Yuan-Tsong Chen You-Di Liao 《The Journal of biological chemistry》2010,285(12):8985-8994
Cationic antimicrobial peptides/proteins (AMPs) are important components of the host innate defense mechanisms against invading microorganisms. Here we demonstrate that OprI (outer membrane protein I) of Pseudomonas aeruginosa is responsible for its susceptibility to human ribonuclease 7 (hRNase 7) and α-helical cationic AMPs, instead of surface lipopolysaccharide, which is the initial binding site of cationic AMPs. The antimicrobial activities of hRNase 7 and α-helical cationic AMPs against P. aeruginosa were inhibited by the addition of exogenous OprI or anti-OprI antibody. On modification and internalization of OprI by hRNase 7 into cytosol, the bacterial membrane became permeable to metabolites. The lipoprotein was predicted to consist of an extended loop at the N terminus for hRNase 7/lipopolysaccharide binding, a trimeric α-helix, and a lysine residue at the C terminus for cell wall anchoring. Our findings highlight a novel mechanism of antimicrobial activity and document a previously unexplored target of α-helical cationic AMPs, which may be used for screening drugs to treat antibiotic-resistant bacterial infection. 相似文献
17.
Yoshinobu Kariya Chihiro Kawamura Toshiki Tabei Jianguo Gu 《The Journal of biological chemistry》2010,285(5):3330-3340
Laminin-332 (Lm332; formerly laminin-5) is a basement membrane protein in the skin, which promotes cell motility in wound healing and cancer invasion. In a previous study, we reported that the introduction of bisecting GlcNAc into Lm332 (GnT-III-Lm332), catalyzed by N-acetylglucosaminyltransferase III (GnT-III), reduced cell migration (Kariya, Y., Kato, R., Itoh, S., Fukuda, T., Shibukawa, Y., Sanzen, N., Sekiguchi, K., Wada, Y., Kawasaki, N., and Gu, J. (2008) J. Biol. Chem. 283, 33036–33045). However, the underlying molecular mechanism by which GnT-III-Lm332 suppresses the normal biological functions of Lm332 remains to be elucidated. In this study, we show that galectin-3, which is a β-galactoside-binding protein, strongly bound to unmodified Lm332 but not to GnT-III-Lm332 and that binding of galectin-3 was completely blocked by lactose. Exogenous galectin-3 significantly enhanced keratinocyte cell motility on control Lm332 but not on GnT-III-Lm332. A functional blocking antibody against galectin-3 inhibited Lm332-induced α3β1 and α6β4 integrin clustering and focal contact formation. Co-immunoprecipitation revealed that galectin-3 associated with both β4 integrin and epidermal growth factor receptor, thereby cross-linking the two molecules. The associations were inhibited by either the presence of lactose or expression of GnT-III. Moreover, galectin-3 consistently enhanced ERK activation. Taken together, the results of this study are the first to clearly identify the molecular mechanism responsible for the inhibitory effects of GnT-III on extracellular matrix-integrin-meditated cell adhesion, migration, and signal transduction. The findings presented herein shed light on the importance of N-glycosylation-mediated supramolecular complex formation on the cell surface. 相似文献
18.
Kitadokoro K Nishimura K Kamitani S Fukui-Miyazaki A Toshima H Abe H Kamata Y Sugita-Konishi Y Yamamoto S Karatani H Horiguchi Y 《The Journal of biological chemistry》2011,286(22):19549-19555
Clostridium perfringens enterotoxin (CPE) is a cause of food poisoning and is considered a pore-forming toxin, which damages target cells by disrupting the selective permeability of the plasma membrane. However, the pore-forming mechanism and the structural characteristics of the pores are not well documented. Here, we present the structure of CPE determined by x-ray crystallography at 2.0 Å. The overall structure of CPE displays an elongated shape, composed of three distinct domains, I, II, and III. Domain I corresponds to the region that was formerly referred to as C-CPE, which is responsible for binding to the specific receptor claudin. Domains II and III comprise a characteristic module, which resembles those of β-pore-forming toxins such as aerolysin, C. perfringens ϵ-toxin, and Laetiporus sulfureus hemolytic pore-forming lectin. The module is mainly made up of β-strands, two of which span its entire length. Domain II and domain III have three short β-strands each, by which they are distinguished. In addition, domain II has an α-helix lying on the β-strands. The sequence of amino acids composing the α-helix and preceding β-strand demonstrates an alternating pattern of hydrophobic residues that is characteristic of transmembrane domains forming β-barrel-made pores. These structural features imply that CPE is a β-pore-forming toxin. We also hypothesize that the transmembrane domain is inserted into the membrane upon the buckling of the two long β-strands spanning the module, a mechanism analogous to that of the cholesterol-dependent cytolysins. 相似文献
19.
Lee HH 《Molecules and cells》2012,33(3):229-233
Shikimate dehydrogenase (SDH), which catalyses the NADPH-dependent reduction of 3-dehydroshikimate to shikimate in the shikimate pathway, is an attractive target for the development of herbicides and antimicrobial agents. Structural analysis of a SDH from Thermotoga maritima encoded by the Tm0346 gene was performed to facilitate further structural comparisons between the various shikimate dehydrogenases. The crystal structure of SDH from T. maritima was determined at 1.45 Å by molecular replacement. SDH from T. maritima showed a monomeric architecture. The overall structure of SDH from T. maritima comprises the N-terminal α/β sandwich domain for substrate binding and the C-terminal domain for NADP binding. When the T. maritima SDH structure was compared with those of the SDHs from other species, the SDH from T. maritima was in a tightly closed conformation, which should be open for catalysis. Notably, α7 moves toward the active site (∼5 Å), which forces the SDH of T. maritima in a more closed form. Four ammonium sulfate (AMS) ions were identified in the structure. They were located in the active site and appeared to mimic the role of the substrate in terms of the enzyme activity and stability. The new high resolution structural information reported in this study, including the AMS binding sites as a potent inhibitor binding site of SDHs, is expected to supplement the existing structural data and will be useful for structure-based antibacterial discovery against SDHs. 相似文献