12 results on '"Cowieson, Nathan"'
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2. Time-resolved studies of dynamic biomolecules using small angle X-ray scattering.
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Kirby, Nigel M and Cowieson, Nathan P
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X-ray scattering , *BIOMOLECULES , *TIME-resolved spectroscopy , *BIOMACROMOLECULES , *MOLECULAR structure , *CHROMATOGRAPHIC analysis - Abstract
Small angle X-ray scattering (SAXS) of biomacromolecules in solution has become a prominent technique in structural biology. Whilst the majority of current use is for static measurements, the field is also advancing for measurements where the sample at the beam position changes with time, using high throughput systems, chromatography, high speed mixing and pump-probe techniques in particular. Time resolved work is greatly aided by increasingly sophisticated software for acquiring and analysing data, together with developments in X-ray sources, beamline optics and detectors. The exploitation of spatial coherence is under development, with X-ray free electron lasers aiming to provide major advances in single molecule structure reconstruction and time resolution. Here we provide an overview of current developments advancing time resolved solution SAXS. [ABSTRACT FROM AUTHOR]
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- 2014
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3. The X-ray Crystal Structure of Full-Length Human Plasminogen
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Law, Ruby H.P., Caradoc-Davies, Tom, Cowieson, Nathan, Horvath, Anita J., Quek, Adam J., Encarnacao, Joanna Amarante, Steer, David, Cowan, Angus, Zhang, Qingwei, Lu, Bernadine G.C., Pike, Robert N., Smith, A. Ian, Coughlin, Paul B., and Whisstock, James C.
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PLASMINOGEN ,PROTEIN precursors ,FIBRINOLYTIC agents ,SERINE proteinases ,UROKINASE ,GLYCOSYLATION ,CRYSTAL structure - Abstract
Summary: Plasminogen is the proenzyme precursor of the primary fibrinolytic protease plasmin. Circulating plasminogen, which comprises a Pan-apple (PAp) domain, five kringle domains (KR1-5), and a serine protease (SP) domain, adopts a closed, activation-resistant conformation. The kringle domains mediate interactions with fibrin clots and cell-surface receptors. These interactions trigger plasminogen to adopt an open form that can be cleaved and converted to plasmin by tissue-type and urokinase-type plasminogen activators. Here, the structure of closed plasminogen reveals that the PAp and SP domains, together with chloride ions, maintain the closed conformation through interactions with the kringle array. Differences in glycosylation alter the position of KR3, although in all structures the loop cleaved by plasminogen activators is inaccessible. The ligand-binding site of KR1 is exposed and likely governs proenzyme recruitment to targets. Furthermore, analysis of our structure suggests that KR5 peeling away from the PAp domain may initiate plasminogen conformational change. [ABSTRACT FROM AUTHOR]
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- 2012
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4. United we stand: combining structural methods
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Cowieson, Nathan P, Kobe, Bostjan, and Martin, Jennifer L
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MOLECULAR biology techniques , *BIOLOGICAL systems , *MEMBRANE proteins , *BIOLOGICAL membranes , *PROTEINS - Abstract
High-resolution techniques are the mainstay of structural biologists; however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins. [Copyright &y& Elsevier]
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- 2008
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5. Cortactin Adopts a Globular Conformation and Bundles Actin into Sheets.
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Cowieson, Nathan P., King, Gordon, Cookson, David, Ross, Ian, Huber, Thomas, Hume, David A., Kobe, Bostjan, and Martin, Jennifer L.
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BIOCHEMISTRY , *ACTIN , *CYTOSKELETON , *PHAGOCYTOSIS , *CELL migration , *CIRCULAR dichroism - Abstract
Cortactin is a filamentous actin-binding protein that plays a pivotal role in translating environmental signals into coordinated rearrangement of the cytoskeleton. The dynamic reorganization of actin in the cytoskeleton drives processes including changes in cell morphology, cell migration, and phagocytosis. In general, structural proteins of the cytoskeleton bind in the N-terminal region of cortactin and regulatory proteins in the C-terminal region. Previous structural studies have reported an extended conformation for cortactin. It is therefore unclear how cortactin facilitates cross-talk between structural proteins and their regulators. In the study presented here, circular dichroism, chemical cross-linking, and small angle x-ray scattering are used to demonstrate that cortactin adopts a globular conformation, thereby bringing distant parts of the molecule into close proximity. In addition, the actin bundling activity of cortactin is characterized, showing that fully polymerized actin filaments are bundled into sheet-like structures. We present a low resolution structure that suggests how the various domains of cortactin interact to coordinate its array of binding partners at sites of actin branching. [ABSTRACT FROM AUTHOR]
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- 2008
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6. Structural Basis for Recognition of High Mannose Type Glycoproteins by Mammalian Transport Lectin VlP36.
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Satoh, Tadashi, Cowieson, Nathan P., Hakamata, Wataru, Ideo, Hiroko, Fukushima, Keiko, Kurihara, Masaaki, Kato, Ryuichi, Yamashita, Katsuko, and Wakatsuki, Soichi
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MANNOSE , *MONOSACCHARIDES , *GLYCOPROTEINS , *LECTINS , *HEMAGGLUTININ , *HYDROGEN bonding , *PROTEIN binding - Abstract
VIP36 functions as a transport lectin for trafficking certain high man nose type glycoproteins in the secretory pathway. Here we report the crystal structure of VIP36 exoplasmic/luminal domain comprising a carbohydrate recognition domain and a stalk domain. The structures of VIP36 in complex with Ca2+ and mannosyl ligands are also described. The carbohydrate recognition domain is composed of a 17-stranded antiparallel a-sandwich and binds one Ca2+ adjoining the carbohydrate-binding site. The structure reveals that a coordinated Ca2+ ion orients the side chains of Asp131, Asn166, and His190 for carbohydrate binding. This result explains the Ca2+-dependent carbohydrate binding of this protein. The Man-α-1,2-Man-α-1,2-Man, which corresponds to the D1 arm of high mannose type glycan, is recognized by eight residues through extensive hydrogen bonds. The complex structures reveal the structural basis for high man-nose type glycoprotein recognition by VIP36 in a Ca2+dependent and D1 arm-specific manner. [ABSTRACT FROM AUTHOR]
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- 2007
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7. Structural comparison of typical and atypical E2 pestivirus glycoproteins.
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Aitkenhead, Hazel, Riedel, Christiane, Cowieson, Nathan, Rümenapf, Hans Tillmann, Stuart, David I., and El Omari, Kamel
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GLYCOPROTEINS , *RATTUS norvegicus , *MEMBRANE glycoproteins , *VETERINARY virology , *CRYSTAL structure - Abstract
Pestiviruses, within the family Flaviviridae , are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed β-hairpin reported to be involved in host receptor binding. Functional analysis of this β-hairpin in the context of BVDV revealed its ability to rescue viral infectivity. [Display omitted] • The X-ray crystal structures of APPV and NRPV E2 glycoproteins were determined • APPV and NRPV E2 form a dimer in solution Aitkenhead et al. report the X-ray crystal structures of pestivirus E2 surface glycoproteins from atypical porcine pestivirus and Norway rat pestivirus. These glycoproteins serve as primary targets for neutralizing antibodies and play crucial roles in cell attachment and viral fusion. [ABSTRACT FROM AUTHOR]
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- 2024
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8. Structural insights into GDP-mediated regulation of a bacterial acyl-CoA thioesterase.
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Khandokar, Yogesh B., Srivastava, Parul, Cowieson, Nathan, Sarker, Subir, Aragao, David, Das, Shubagata, Smith, Kate M., Raidal, Shane R., and Forwood, Jade K.
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THIOESTERASE , *BIOCHEMICAL substrates , *CATALYTIC activity , *AMINO acids , *ALANINE - Abstract
Thioesterases catalyze the cleavage of thioester bonds within many activated fatty acids and acyl-CoA substrates. They are expressed ubiquitously in both prokaryotes and eukaryotes and are subdivided into 25 thioesterase families according to their catalytic active site, protein oligomerization, and substrate specificity. Although many of these enzyme families are well-characterized in terms of function and substrate specificity, regulation across most thioesterase families is poorly understood. Here, we characterized a TE6 thioesterase from the bacterium Neisseria meningitidis Structural analysis with X-ray crystallographic diffraction data to 2.0-Å revealed that each protein subunit harbors a hot dog-fold and that the TE6 enzyme forms a hexamer with D3 symmetry. An assessment of thioesterase activity against a range of acyl-CoA substrates revealed the greatest activity against acetyl-CoA, and structure-guided mutagenesis of putative active site residues identified Asn24 and Asp39 as being essential for activity. Our structural analysis revealed that six GDP nucleotides bound the enzyme in close proximity to an intersubunit disulfide bond interactions that covalently link thioesterase domains in a double hot dog dimer. Structure-guided mutagenesis of residues within the GDP-binding pocket identified Arg93 as playing a key role in the nucleotide interaction and revealed that GDP is required for activity. All mutations were confirmed to be specific and not to have resulted from structural perturbations by X-ray crystallography. This is the first report of a bacterial GDP-regulated thioesterase and of covalent linkage of thioesterase domains through a disulfide bond, revealing structural similarities with ADP regulation in the human ACOT12 thioesterase. [ABSTRACT FROM AUTHOR]
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- 2017
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9. Structural Basis for Regulation of the Human Acetyl-CoA Thioesterase 12 and Interactions with the Steroidogenic Acute Regulatory Protein-related Lipid Transfer (START) Domain.
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Swarbrick, Crystall M. D., Roman, Noelia, Cowieson, Nathan, Patterson, Edward I., Nanson, Jeffrey, Siponen, Marina I., Berglund, Helena, Lehtiö, Lari, and Forwood, Jade K.
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ACETYLCOENZYME A , *ACETYL-CoA synthetase , *GENETIC regulation , *THIOESTERASE , *PROTEIN expression , *STEROIDOGENIC acute regulatory protein - Abstract
Acetyl-CoA plays a fundamental role in cell signaling and metabolic pathways, with its cellular levels tightly controlled through reciprocal regulation of enzymes that mediate its synthesis and catabolism. ACOT12, the primary acetyl-CoA thioesterase in the liver of human, mouse, and rat, is responsible for cleavage of the thioester bond within acetyl-CoA, producing acetate and coenzyme A for a range of cellular processes. The enzyme is regulated by ADP and ATP, which is believed to be mediated through the ligand-induced oligomerization of the thioesterase domains, whereby ATP induces active dimers and tetramers, whereas apo- and ADP-bound ACOT12 are monomeric and inactive. Here, using a range of structural and biophysical techniques, it is demonstrated that ACOT12 is a trimer rather than a tetramer and that neitherADPnorATPexert their regulatory effects by altering the oligomeric status of the enzyme. Rather, the binding site and mechanism of ADP regulation have been determined to occur through two novel regulatory regions, one involving a large loop that links the thioesterase domains (Phe154-Thr178), defined here as RegLoop1, and a second region involving theCterminus of thioesterase domain 2 (Gln304-Gly326), designated RegLoop2. Mutagenesis confirmed that Arg312 and Arg313 are crucial for this mode of regulation, and novel interactions with the START domain are presented together with insights into domain swapping within eukaryotic thioesterases for substrate recognition. In summary, these experiments provide the first structural insights into the regulation of this enzyme family, revealing an alternate hypothesis likely to be conserved throughout evolution. [ABSTRACT FROM AUTHOR]
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- 2014
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10. An Inflammatory Role for the Mammalian Carboxypeptidase Inhibitor Latexin: Relationship to Cystatins and the Tumor Suppressor TIG1
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Aagaard, Anna, Listwan, Pawel, Cowieson, Nathan, Huber, Thomas, Ravasi, Timothy, Wells, Christine A., Flanagan, Jack U., Kellie, Stuart, Hume, David A., Kobe, Bostjan, and Martin, Jennifer L.
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CARBOXYPEPTIDASES , *PROTEINS , *TUMORS , *INFLAMMATION - Abstract
Summary: Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Å resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface. [Copyright &y& Elsevier]
- Published
- 2005
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11. Structural and Functional Characterization of the PaaI Thioesterase from Streptococcus pneumoniae Reveals a Dual Specificity for Phenylacetyl-CoA and Medium-chain Fatty Acyl-CoAs and a Novel CoA-induced Fit Mechanism.
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Khandokar, Yogesh B., Srivastava, Parul, Sarker, Subir, Swarbrick, Crystall M. D., Aragao, David, Cowieson, Nathan, and Forwood, Jade K.
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STREPTOCOCCUS pneumoniae , *THIOESTERASE , *FATTY-acyl-CoA , *HYDROLYSIS , *CRYSTALLOGRAPHY , *GEL permeation chromatography - Abstract
PaaI thioesterases are members of the TE13 thioesterase family that catalyze the hydrolysis of thioester bonds between coenzyme A and phenylacetyl-CoA. In this study we characterize the PaaI thioesterase from Streptococcus pneumoniae (SpPaaI), including structural analysis based on crystal diffraction data to 1.8-Å resolution, to reveal two double hotdog domains arranged in a back to back configuration. Consistent with the crystallography data, both size exclusion chromatography and small angle x-ray scattering data support a tetrameric arrangement of thioesterase domains in solution. Assessment of SpPaaI activity against a range of acyl-CoA substrates showed activity for both phenylacetyl-CoA and medium-chain fatty-acyl CoA substrates. Mutagenesis of putative active site residues reveals Asn37, Asp52, and Thr68 are important for catalysis, and size exclusion chromatography analysis and x-ray crystallography confirm that these mutants retain the same tertiary and quaternary structures, establishing that the reduced activity is not a result of structural perturbations. Interestingly, the structure of SpPaaI in the presence of CoA provides a structural basis for the observed substrate specificity, accommodating a 10-carbon fatty acid chain, and a large conformational change of up to 38Å in theNterminus, and a loop region involving Tyr38-Tyr39. This is the first time PaaI thioesterases have displayed a dual specificity for medium-chain acyl-CoAs substrates and phenylacetyl-CoA substrates, and we provide a structural basis for this specificity, highlighting a novel induced fit mechanism that is likely to be conserved within members of this enzyme family. [ABSTRACT FROM AUTHOR]
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- 2016
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12. Dimerization of Plant Defensin NaD1 Enhances Its Antifungal Activity.
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Lay, Fung T., Mills, Grant D., Poon, Ivan K. H., Cowieson, Nathan P., Kirby, Nigel, Baxter, Amy A., van der Weerden, Nicole L., Dogovski, Con, Perugini, Matthew A., Anderson, Marilyn A., Kvansakul, Marc, and Hulett, Mark D.
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DIMERIZATION , *DEFENSINS , *NICOTIANA , *ANTIFUNGAL agents , *FUSARIUM oxysporum , *ULTRACENTRIFUGATION , *PLANT protection - Abstract
The plant defensin, NaD1, from the flowers of Nicotiana alata, is a member of a family of cationic peptides that displays growth inhibitory activity against several filamentous fungi, including Fusarium oxysporum. The antifungal activity of NaD1 has been attributed to its ability to permeabilize membranes; however, the molecular basis of this function remains poorly defined. In this study, we have solved the structure of NaD1 from two crystal forms to high resolution (1.4 and 1.58 Å, respectively), both of which contain NaD1 in a dimeric configuration. Using protein cross-linking experiments as well as small angle x-ray scattering analysis and analytical ultracentrifugation, we show that NaD1 forms dimers in solution. The structural studies identified Lys4 as critical in formation of the NaD1 dimer. This was confirmed by site-directed mutagenesis of Lys4 that resulted in substantially reduced dimer formation. Significantly, the reduced ability of the Lys4 mutant to dimerize correlated with diminished antifungal activity. These data demonstrate the importance of dimerization in NaD1 function and have implications for the use of defensins in agribiotechnology applications such as enhancing plant crop protection against fungal pathogens. [ABSTRACT FROM AUTHOR]
- Published
- 2012
- Full Text
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