63 results on '"Harrop SJ"'
Search Results
2. CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: A smoking gun?
- Author
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Jiang, L, Phang, JM, Yu, J, Harrop, SJ, Sokolova, AV, Duff, AP, Wilk, KE, Alkhamici, H, Breit, SN, Valenzuela, SM, Brown, LJ, Curmi, PMG, Jiang, L, Phang, JM, Yu, J, Harrop, SJ, Sokolova, AV, Duff, AP, Wilk, KE, Alkhamici, H, Breit, SN, Valenzuela, SM, Brown, LJ, and Curmi, PMG
- Abstract
The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé. © 2013 Elsevier B.V.
- Published
- 2014
3. A putative house-cleaning enzyme encoded within an integron array: 1.8 angstrom crystal structure defines a new MazG subtype
- Author
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Robinson, A, Guilfoyle, AP, Harrop, SJ, Boucher, Y, Stokes, HW, Curmi, PMG, and Mabbutt, BC
- Subjects
Models, Molecular ,Molecular Structure ,Sequence Homology, Amino Acid ,Models, Genetic ,Molecular Sequence Data ,Crystallography, X-Ray ,Microbiology ,Protein Structure, Secondary ,Integrons ,Protein Structure, Tertiary ,Substrate Specificity ,Bacterial Proteins ,Amino Acid Sequence ,Pyrophosphatases ,Vibrio - Published
- 2007
4. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family
- Author
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Robinson, A, Wu, PS-C, Harrop, SJ, Schaeffer, PM, Dosztányi, Z, Gillings, MR, Holmes, AJ, Nevalainen, KMH, Stokes, HW, Otting, G, Dixon, NE, Curmi, PMG, and Mabbutt, BC
- Subjects
Epoxide Hydrolases ,Models, Molecular ,Biochemistry & Molecular Biology ,Protein Folding ,Ion Transport ,Sequence Homology, Amino Acid ,Molecular Sequence Data ,Crystallography, X-Ray ,Protein Structure, Secondary ,Integrons ,Soil ,Bacterial Proteins ,Catalytic Domain ,DNA Transposable Elements ,Amino Acid Sequence ,Isomerases ,Soil Microbiology - Abstract
The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.
- Published
- 2004
5. Integron Gene Cassettes: A Repository of Novel Protein Folds with Distinct Interaction Sites
- Author
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Sureshan, V, Deshpande, CN, Boucher, Y, Koenig, JE, Stokes, HW, Harrop, SJ, Curmi, PMG, Mabbutt, BC, Sureshan, V, Deshpande, CN, Boucher, Y, Koenig, JE, Stokes, HW, Harrop, SJ, Curmi, PMG, and Mabbutt, BC
- Published
- 2013
6. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module
- Author
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Deshpande, CN, Harrop, SJ, Boucher, Y, Hassan, KA, Leo, RD, Xu, X, Cui, H, Savchenko, A, Chang, C, Labbate, M, Paulsen, IT, Stokes, HW, Curmi, PMG, Mabbutt, BC, Deshpande, CN, Harrop, SJ, Boucher, Y, Hassan, KA, Leo, RD, Xu, X, Cui, H, Savchenko, A, Chang, C, Labbate, M, Paulsen, IT, Stokes, HW, Curmi, PMG, and Mabbutt, BC
- Abstract
Background: The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings: We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance: Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds. © 2011 Deshpande et al.
- Published
- 2011
7. The enigma of the CLIC proteins: Ion channels, redox proteins, enzymes, scaffolding proteins?
- Author
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Littler, DR, Harrop, SJ, Goodchild, SC, Phang, JM, Mynott, AV, Jiang, L, Valenzuela, SM, Mazzanti, M, Brown, LJ, Breit, SN, Curmi, PMG, Littler, DR, Harrop, SJ, Goodchild, SC, Phang, JM, Mynott, AV, Jiang, L, Valenzuela, SM, Mazzanti, M, Brown, LJ, Breit, SN, and Curmi, PMG
- Abstract
Chloride intracellular channel proteins (CLICs) are distinct from most ion channels in that they have both soluble and integral membrane forms. CLICs are highly conserved in chordates, with six vertebrate paralogues. CLIC-like proteins are found in other metazoans. CLICs form channels in artificial bilayers in a process favoured by oxidising conditions and low pH. They are structurally plastic, with CLIC1 adopting two distinct soluble conformations. Phylogenetic and structural data indicate that CLICs are likely to have enzymatic function. The physiological role of CLICs appears to be maintenance of intracellular membranes, which is associated with tubulogenesis but may involve other substructures. © 2010 Federation of European Biochemical Societies.
- Published
- 2010
8. Structural Genomics of the Bacterial Mobile Metagenome: an Overview
- Author
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Kobe, B, Guss, M, Huber, T, Robinson, A, Guilfoyle, AP, Sureshan, V, Howell, M, Harrop, SJ, Boucher, Y, Stokes, H, Curmi, PM, Mabbutt, BC, Kobe, B, Guss, M, Huber, T, Robinson, A, Guilfoyle, AP, Sureshan, V, Howell, M, Harrop, SJ, Boucher, Y, Stokes, H, Curmi, PM, and Mabbutt, BC
- Published
- 2008
9. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.
- Author
-
Robinson, A, Wu, PS-C, Harrop, SJ, Schaeffer, PM, Dosztányi, Z, Gillings, MR, Holmes, AJ, Nevalainen, KMH, Stokes, HW, Otting, G, Dixon, NE, Curmi, PMG, Mabbutt, BC, Robinson, A, Wu, PS-C, Harrop, SJ, Schaeffer, PM, Dosztányi, Z, Gillings, MR, Holmes, AJ, Nevalainen, KMH, Stokes, HW, Otting, G, Dixon, NE, Curmi, PMG, and Mabbutt, BC
- Abstract
The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.
- Published
- 2005
10. Molecular structures reveal the origin of spectral variation in cryptophyte light harvesting antenna proteins.
- Author
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Michie KA, Harrop SJ, Rathbone HW, Wilk KE, Teng CY, Hoef-Emden K, Hiller RG, Green BR, and Curmi PMG
- Subjects
- Molecular Structure, Chlorophyll A metabolism, Models, Molecular, Amino Acid Sequence, Cryptophyta metabolism
- Abstract
In addition to their membrane-bound chlorophyll a/c light-harvesting antenna, the cryptophyte algae have evolved a unique phycobiliprotein antenna system located in the thylakoid lumen. The basic unit of this antenna consists of two copies of an αβ protomer where the α and β subunits scaffold different combinations of a limited number of linear tetrapyrrole chromophores. While the β subunit is highly conserved, encoded by a single plastid gene, the nuclear-encoded α subunits have evolved diversified multigene families. It is still unclear how this sequence diversity results in the spectral diversity of the mature proteins. By careful examination of three newly determined crystal structures in comparison with three previously obtained, we show how the α subunit amino acid sequences control chromophore conformations and hence spectral properties even when the chromophores are identical. Previously we have shown that α subunits control the quaternary structure of the mature αβ.αβ complex (either open or closed), however, each species appeared to only harbor a single quaternary form. Here we show that species of the Hemiselmis genus contain expressed α subunit genes that encode both distinct quaternary structures. Finally, we have discovered a common single-copy gene (expressed into protein) consisting of tandem copies of a small α subunit that could potentially scaffold pairs of light harvesting units. Together, our results show how the diversity of the multigene α subunit family produces a range of mature cryptophyte antenna proteins with differing spectral properties, and the potential for minor forms that could contribute to acclimation to varying light regimes., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)
- Published
- 2023
- Full Text
- View/download PDF
11. Crystal structure of a UDP-GlcNAc epimerase for surface polysaccharide biosynthesis in Acinetobacter baumannii.
- Author
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Shah BS, Ashwood HE, Harrop SJ, Farrugia DN, Paulsen IT, and Mabbutt BC
- Subjects
- Acinetobacter baumannii genetics, Acinetobacter baumannii isolation & purification, Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Carbohydrate Epimerases genetics, Carbohydrate Epimerases metabolism, Catalytic Domain, Crystallography, X-Ray, Humans, Models, Molecular, Polysaccharides, Bacterial biosynthesis, Protein Conformation, Protein Domains, Protein Structure, Quaternary, Sequence Homology, Amino Acid, Static Electricity, Structural Homology, Protein, Acinetobacter baumannii enzymology, Bacterial Proteins chemistry, Carbohydrate Epimerases chemistry
- Abstract
With new strains of Acinetobacter baumannii undergoing genomic analysis, it has been possible to define regions of genomic plasticity (RGPs), encoding specific adaptive elements. For a selected RGP from a community-derived isolate of A. baumannii, we outline sequences compatible with biosynthetic machinery of surface polysaccharides, specifically enzymes utilized in the dehydration and conversion of UDP-N-acetyl-D-glucosamine (UDP-D-GlcNAc). We have determined the crystal structure of one of these, the epimerase Ab-WbjB. This dehydratase belongs to the 'extended' short-chain dehydrogenase/reductase (SDR) family, related in fold to previously characterised enzymes CapE and FlaA1. Our 2.65Å resolution structure of Ab-WbjB shows a hexamer, organised into a trimer of chain pairs, with coenzyme NADP+ occupying each chain. Specific active-site interactions between each coenzyme and a lysine quaternary group of a neighbouring chain interconnect adjacent dimers, so stabilising the hexameric form. We show UDP-GlcNAc to be a specific substrate for Ab-WbjB, with binding evident by ITC (Ka = 0.23 μmol-1). The sequence of Ab-WbjB shows variation from the consensus active-site motifs of many SDR enzymes, demonstrating a likely catalytic role for a specific threonine sidechain (as an alternative to tyrosine) in the canonical active site chemistry of these epimerases.
- Published
- 2018
- Full Text
- View/download PDF
12. Structural characterization suggests models for monomeric and dimeric forms of full-length ezrin.
- Author
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Phang JM, Harrop SJ, Duff AP, Sokolova AV, Crossett B, Walsh JC, Beckham SA, Nguyen CD, Davies RB, Glöckner C, Bromley EH, Wilk KE, and Curmi PM
- Subjects
- Circular Dichroism, Crystallography, X-Ray, Dimerization, Protein Conformation, Cytoskeletal Proteins chemistry
- Abstract
Ezrin is a member of the ERM (ezrin-radixin-moesin) family of proteins that have been conserved through metazoan evolution. These proteins have dormant and active forms, where the latter links the actin cytoskeleton to membranes. ERM proteins have three domains: an N-terminal FERM [band Four-point-one (4.1) ERM] domain comprising three subdomains (F1, F2, and F3); a helical domain; and a C-terminal actin-binding domain. In the dormant form, FERM and C-terminal domains form a stable complex. We have determined crystal structures of the active FERM domain and the dormant FERM:C-terminal domain complex of human ezrin. We observe a bistable array of phenylalanine residues in the core of subdomain F3 that is mobile in the active form and locked in the dormant form. As subdomain F3 is pivotal in binding membrane proteins and phospholipids, these transitions may facilitate activation and signaling. Full-length ezrin forms stable monomers and dimers. We used small-angle X-ray scattering to determine the solution structures of these species. As expected, the monomer shows a globular domain with a protruding helical coiled coil. The dimer shows an elongated dumbbell structure that is twice as long as the monomer. By aligning ERM sequences spanning metazoan evolution, we show that the central helical region is conserved, preserving the heptad repeat. Using this, we have built a dimer model where each monomer forms half of an elongated antiparallel coiled coil with domain-swapped FERM:C-terminal domain complexes at each end. The model suggests that ERM dimers may bind to actin in a parallel fashion., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)
- Published
- 2016
- Full Text
- View/download PDF
13. Structure of the PLP-Form of the Human Kynurenine Aminotransferase II in a Novel Spacegroup at 1.83 Å Resolution.
- Author
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Nematollahi A, Sun G, Harrop SJ, Hanrahan JR, and Church WB
- Subjects
- Binding Sites, Catalytic Domain, Crystallography, X-Ray, Humans, Hydrogen Bonding, Protein Structure, Tertiary, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Transaminases genetics, Transaminases metabolism, Transaminases chemistry
- Abstract
Kynurenine aminotransferase II (KAT-II) is a 47 kDa pyridoxal phosphate (PLP)-dependent enzyme, active as a homodimer, which catalyses the transamination of the amino acids kynurenine (KYN) and 3-hydroxykynurenine (3-HK) in the tryptophan pathway, and is responsible for producing metabolites that lead to kynurenic acid (KYNA), which is implicated in several neurological diseases such as schizophrenia. In order to fully describe the role of KAT-II in the pathobiology of schizophrenia and other brain disorders, the crystal structure of full-length PLP-form hKAT-II was determined at 1.83 Å resolution, the highest available. The electron density of the active site reveals an aldimine linkage between PLP and Lys263, as well as the active site residues, which characterize the fold-type I PLP-dependent enzymes.
- Published
- 2016
- Full Text
- View/download PDF
14. Structure of GUN4 from Chlamydomonas reinhardtii.
- Author
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Tarahi Tabrizi S, Langley DB, Harrop SJ, Duff AP, and Willows RD
- Subjects
- Algal Proteins genetics, Amino Acid Sequence, Base Sequence, Binding Sites, Chlamydomonas reinhardtii genetics, Cloning, Molecular, Crystallization, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Folding, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Fusion Proteins genetics, Sequence Alignment, Algal Proteins chemistry, Chlamydomonas reinhardtii chemistry, Protoporphyrins chemistry, Recombinant Fusion Proteins chemistry
- Abstract
The genomes uncoupled 4 (GUN4) protein stimulates chlorophyll biosynthesis by increasing the activity of Mg-chelatase, the enzyme that inserts magnesium into protoporphyrin IX (PPIX) in the chlorophyll biosynthesis pathway. One of the roles of GUN4 is in binding PPIX and Mg-PPIX. In eukaryotes, GUN4 also participates in plastid-to-nucleus signalling, although the mechanism for this is unclear. Here, the first crystal structure of a eukaryotic GUN4, from Chlamydomonas reinhardtii, is presented. The structure is in broad agreement with those of previously solved cyanobacterial structures. Most interestingly, conformational divergence is restricted to several loops which cover the porphyrin-binding cleft. The conformational dynamics suggested by this ensemble of structures lend support to the understanding of how GUN4 binds PPIX or Mg-PPIX.
- Published
- 2015
- Full Text
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15. MX1: a bending-magnet crystallography beamline serving both chemical and macromolecular crystallography communities at the Australian Synchrotron.
- Author
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Cowieson NP, Aragao D, Clift M, Ericsson DJ, Gee C, Harrop SJ, Mudie N, Panjikar S, Price JR, Riboldi-Tunnicliffe A, Williamson R, and Caradoc-Davies T
- Abstract
MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented.
- Published
- 2015
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- View/download PDF
16. Structural insights into the organization of the cavin membrane coat complex.
- Author
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Kovtun O, Tillu VA, Jung W, Leneva N, Ariotti N, Chaudhary N, Mandyam RA, Ferguson C, Morgan GP, Johnston WA, Harrop SJ, Alexandrov K, Parton RG, and Collins BM
- Subjects
- Amino Acid Sequence, Animals, Caveolae ultrastructure, Crystallography, X-Ray, Cytoplasm chemistry, Cytoplasm ultrastructure, Membrane Proteins metabolism, Microscopy, Electron, Molecular Sequence Data, Protein Structure, Quaternary, Signal Transduction physiology, Zebrafish metabolism, Caveolae chemistry, Caveolae metabolism, Caveolins chemistry, Caveolins metabolism, Cytoplasm metabolism, RNA-Binding Proteins chemistry, RNA-Binding Proteins metabolism
- Abstract
Caveolae are cell-surface membrane invaginations that play critical roles in cellular processes including signaling and membrane homeostasis. The cavin proteins, in cooperation with caveolins, are essential for caveola formation. Here we show that a minimal N-terminal domain of the cavins, termed HR1, is required and sufficient for their homo- and hetero-oligomerization. Crystal structures of the mouse cavin1 and zebrafish cavin4a HR1 domains reveal highly conserved trimeric coiled-coil architectures, with intersubunit interactions that determine the specificity of cavin-cavin interactions. The HR1 domain contains a basic surface patch that interacts with polyphosphoinositides and coordinates with additional membrane-binding sites within the cavin C terminus to facilitate membrane association and remodeling. Electron microscopy of purified cavins reveals the existence of large assemblies, composed of a repeating rod-like structural element, and we propose that these structures polymerize through membrane-coupled interactions to form the unique striations observed on the surface of caveolae in vivo., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
17. Structure of a short-chain dehydrogenase/reductase (SDR) within a genomic island from a clinical strain of Acinetobacter baumannii.
- Author
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Shah BS, Tetu SG, Harrop SJ, Paulsen IT, and Mabbutt BC
- Subjects
- Acinetobacter baumannii genetics, Amino Acid Sequence, Apoenzymes chemistry, Catalytic Domain, Crystallography, X-Ray, Genome, Bacterial, Genomic Islands, Models, Molecular, Molecular Sequence Data, Acinetobacter baumannii enzymology, Bacterial Proteins chemistry, Fatty Acid Synthases chemistry, NADH, NADPH Oxidoreductases chemistry
- Abstract
Over 15% of the genome of an Australian clinical isolate of Acinetobacter baumannii occurs within genomic islands. An uncharacterized protein encoded within one island feature common to this and other International Clone II strains has been studied by X-ray crystallography. The 2.4 Å resolution structure of SDR-WM99c reveals it to be a new member of the classical short-chain dehydrogenase/reductase (SDR) superfamily. The enzyme contains a nucleotide-binding domain and, like many other SDRs, is tetrameric in form. The active site contains a catalytic tetrad (Asn117, Ser146, Tyr159 and Lys163) and water molecules occupying the presumed NADP cofactor-binding pocket. An adjacent cleft is capped by a relatively mobile helical subdomain, which is well positioned to control substrate access.
- Published
- 2014
- Full Text
- View/download PDF
18. Single-residue insertion switches the quaternary structure and exciton states of cryptophyte light-harvesting proteins.
- Author
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Harrop SJ, Wilk KE, Dinshaw R, Collini E, Mirkovic T, Teng CY, Oblinsky DG, Green BR, Hoef-Emden K, Hiller RG, Scholes GD, and Curmi PM
- Subjects
- Amino Acid Sequence, Base Sequence, Crystallography, X-Ray, Dimerization, Molecular Sequence Data, Phycobiliproteins chemistry, Protein Conformation, Sequence Analysis, DNA, Spectrum Analysis, Cryptophyta genetics, Evolution, Molecular, Models, Molecular, Mutagenesis, Insertional genetics, Phycobiliproteins genetics
- Abstract
Observation of coherent oscillations in the 2D electronic spectra (2D ES) of photosynthetic proteins has led researchers to ask whether nontrivial quantum phenomena are biologically significant. Coherent oscillations have been reported for the soluble light-harvesting phycobiliprotein (PBP) antenna isolated from cryptophyte algae. To probe the link between spectral properties and protein structure, we determined crystal structures of three PBP light-harvesting complexes isolated from different species. Each PBP is a dimer of αβ subunits in which the structure of the αβ monomer is conserved. However, we discovered two dramatically distinct quaternary conformations, one of which is specific to the genus Hemiselmis. Because of steric effects emerging from the insertion of a single amino acid, the two αβ monomers are rotated by ∼73° to an "open" configuration in contrast to the "closed" configuration of other cryptophyte PBPs. This structural change is significant for the light-harvesting function because it disrupts the strong excitonic coupling between two central chromophores in the closed form. The 2D ES show marked cross-peak oscillations assigned to electronic and vibrational coherences in the closed-form PC645. However, such features appear to be reduced, or perhaps absent, in the open structures. Thus cryptophytes have evolved a structural switch controlled by an amino acid insertion to modulate excitonic interactions and therefore the mechanisms used for light harvesting.
- Published
- 2014
- Full Text
- View/download PDF
19. CLIC proteins, ezrin, radixin, moesin and the coupling of membranes to the actin cytoskeleton: a smoking gun?
- Author
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Jiang L, Phang JM, Yu J, Harrop SJ, Sokolova AV, Duff AP, Wilk KE, Alkhamici H, Breit SN, Valenzuela SM, Brown LJ, and Curmi PM
- Subjects
- Animals, Humans, Actin Cytoskeleton metabolism, Cell Membrane metabolism, Cytoskeletal Proteins metabolism, Membrane Proteins metabolism, Microfilament Proteins metabolism
- Abstract
The CLIC proteins are a highly conserved family of metazoan proteins with the unusual ability to adopt both soluble and integral membrane forms. The physiological functions of CLIC proteins may include enzymatic activity in the soluble form and anion channel activity in the integral membrane form. CLIC proteins are associated with the ERM proteins: ezrin, radixin and moesin. ERM proteins act as cross-linkers between membranes and the cortical actin cytoskeleton. Both CLIC and ERM proteins are controlled by Rho family small GTPases. CLIC proteins, ERM and Rho GTPases act in a concerted manner to control active membrane processes including the maintenance of microvillar structures, phagocytosis and vesicle trafficking. All of these processes involve the interaction of membranes with the underlying cortical actin cytoskeleton. The relationships between Rho GTPases, CLIC proteins, ERM proteins and the membrane:actin cytoskeleton interface are reviewed. Speculative models are proposed involving the formation of localised multi-protein complexes on the membrane surface that assemble via multiple weak interactions. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
20. Selective inhibition of human group IIA-secreted phospholipase A2 (hGIIA) signaling reveals arachidonic acid metabolism is associated with colocalization of hGIIA to vimentin in rheumatoid synoviocytes.
- Author
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Lee LK, Bryant KJ, Bouveret R, Lei PW, Duff AP, Harrop SJ, Huang EP, Harvey RP, Gelb MH, Gray PP, Curmi PM, Cunningham AM, Church WB, and Scott KF
- Subjects
- Animals, Arachidonic Acid genetics, Arthritis, Rheumatoid drug therapy, Arthritis, Rheumatoid genetics, Arthritis, Rheumatoid pathology, CHO Cells, Cricetinae, Cricetulus, Drug Design, Enzyme Inhibitors therapeutic use, Female, Group II Phospholipases A2 genetics, Group II Phospholipases A2 metabolism, Humans, Male, Signal Transduction genetics, Synovial Membrane pathology, Vimentin genetics, Arachidonic Acid metabolism, Arthritis, Rheumatoid metabolism, Enzyme Inhibitors pharmacology, Group II Phospholipases A2 antagonists & inhibitors, Signal Transduction drug effects, Synovial Membrane metabolism, Vimentin metabolism
- Abstract
Human group IIA secreted phospholipase A2 (hGIIA) promotes tumor growth and inflammation and can act independently of its well described catalytic lipase activity via an alternative poorly understood signaling pathway. With six chemically diverse inhibitors we show that it is possible to selectively inhibit hGIIA signaling over catalysis, and x-ray crystal structures illustrate that signaling involves a pharmacologically distinct surface to the catalytic site. We demonstrate in rheumatoid fibroblast-like synoviocytes that non-catalytic signaling is associated with rapid internalization of the enzyme and colocalization with vimentin. Trafficking of exogenous hGIIA was monitored with immunofluorescence studies, which revealed that vimentin localization is disrupted by inhibitors of signaling that belong to a rare class of small molecule inhibitors that modulate protein-protein interactions. This study provides structural and pharmacological evidence for an association between vimentin, hGIIA, and arachidonic acid metabolism in synovial inflammation, avenues for selective interrogation of hGIIA signaling, and new strategies for therapeutic hGIIA inhibitor design.
- Published
- 2013
- Full Text
- View/download PDF
21. Integron gene cassettes: a repository of novel protein folds with distinct interaction sites.
- Author
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Sureshan V, Deshpande CN, Boucher Y, Koenig JE, Stokes HW, Harrop SJ, Curmi PM, and Mabbutt BC
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Binding Sites, Crystallography, X-Ray, Gene Transfer, Horizontal genetics, Metagenome genetics, Models, Molecular, Molecular Sequence Data, Oligonucleotide Array Sequence Analysis, Protein Binding, Protein Structure, Secondary, Vibrio cholerae genetics, Vibrio cholerae metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Genes, Bacterial genetics, Integrons genetics
- Abstract
Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.
- Published
- 2013
- Full Text
- View/download PDF
22. The RNA polymerase subunits E/F from the Antarctic archaeon Methanococcoides burtonii bind to specific species of mRNA.
- Author
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De Francisci D, Campanaro S, Kornfeld G, Siddiqui KS, Williams TJ, Ertan H, Treu L, Pilak O, Lauro FM, Harrop SJ, Curmi PM, and Cavicchioli R
- Subjects
- Antarctic Regions, Archaeal Proteins genetics, Archaeal Proteins metabolism, Methanosarcinaceae metabolism, Protein Binding, Protein Biosynthesis, RNA, Messenger genetics, Recombinant Proteins metabolism, DNA-Directed RNA Polymerases metabolism, Methanosarcinaceae enzymology, Methanosarcinaceae genetics, RNA, Messenger metabolism
- Abstract
RNA polymerase in Archaea is composed of 11 or 12 subunits - 9 or 10 that form the core, and a heterodimer formed from subunits E and F that associates with the core and can interact with general transcription factors and facilitate transcription. While the ability of the heterodimer to bind RNA has been demonstrated, it has not been determined whether it can recognize specific RNA targets. In this study we used a recombinant archaeal MbRpoE/F to capture cellular mRNA in vitro and a microarray to determine which transcripts it specifically binds. Only transcripts for 117 genes (4% of the total) representing 48 regions of the genome were bound by MbRpoE/F. The transcripts represented important genes in a number of functional classes: methanogenesis, cofactor biosynthesis, nucleotide metabolism, transcription, translation, import/export. The arrangement and characteristics (e.g. codon and amino acid usage) of genes relative to the putative origin of replication indicate that MbRpoE/F preferentially binds to mRNA of genes whose expression may be important for cellular fitness. We also compared the biophysical properties of RpoE/F from M. burtonii and Methanocaldococcus jannaschii, demonstrating a 50°C difference in their apparent melting temperatures. By using MbRpoE/F to capture and characterize cellular RNA we have identified a previously unknown functional property of the MbRpoE/F heterodimer., (© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd.)
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- 2011
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23. Chaperonins from an Antarctic archaeon are predominantly monomeric: crystal structure of an open state monomer.
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Pilak O, Harrop SJ, Siddiqui KS, Chong K, De Francisci D, Burg D, Williams TJ, Cavicchioli R, and Curmi PM
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- Adenosine Triphosphatases metabolism, Amino Acid Sequence, Antarctic Regions, Group II Chaperonins genetics, Group II Chaperonins metabolism, Methanosarcinaceae enzymology, Methanosarcinaceae genetics, Molecular Sequence Data, Phylogeny, Protein Stability, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Sequence Alignment, Temperature, Group II Chaperonins chemistry, Methanosarcinaceae chemistry, Models, Molecular
- Abstract
Archaea are abundant in permanently cold environments. The Antarctic methanogen, Methanococcoides burtonii, has proven an excellent model for studying molecular mechanisms of cold adaptation. Methanococcoides burtonii contains three group II chaperonins that diverged prior to its closest orthologues from mesophilic Methanosarcina spp. The relative abundance of the three chaperonins shows little dependence on organism growth temperature, except at the highest temperatures, where the most thermally stable chaperonin increases in abundance. In vitro and in vivo, the M. burtonii chaperonins are predominantly monomeric, with only 23-33% oligomeric, thereby differing from other archaea where an oligomeric ring form is dominant. The crystal structure of an N-terminally truncated chaperonin reveals a monomeric protein with a fully open nucleotide binding site. When compared with closed state group II chaperonin structures, a large-scale ≈ 30° rotation between the equatorial and intermediate domains is observed resulting in an open nucleotide binding site. This is analogous to the transition observed between open and closed states of group I chaperonins but contrasts with recent archaeal group II chaperonin open state ring structures. The predominance of monomeric form and the ability to adopt a fully open nucleotide site appear to be unique features of the M. burtonii group II chaperonins., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)
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- 2011
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24. Crystal structure of importin-α bound to a peptide bearing the nuclear localisation signal from chloride intracellular channel protein 4.
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Mynott AV, Harrop SJ, Brown LJ, Breit SN, Kobe B, and Curmi PM
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- Active Transport, Cell Nucleus, Animals, Antigens, Viral, Tumor metabolism, Binding Sites, Crystallography, X-Ray, Humans, Mice, Nuclear Localization Signals metabolism, Protein Conformation, Protein Structure, Secondary, Chloride Channels metabolism, alpha Karyopherins metabolism
- Abstract
It has been reported that a human chloride intracellular channel (CLIC) protein, CLIC4, translocates to the nucleus in response to cellular stress, facilitated by a putative CLIC4 nuclear localization signal (NLS). The CLIC4 NLS adopts an α-helical structure in the native CLIC4 fold. It is proposed that CLIC4 is transported to the nucleus via the classical nuclear import pathway after binding the import receptor, importin-α. In this study, we have determined the X-ray crystal structure of a truncated form of importin-α lacking the importin-β binding domain, bound to a CLIC4 NLS peptide. The NLS peptide binds to the major binding site in an extended conformation similar to that observed for the classical simian virus 40 large T-antigen NLS. A Tyr residue within the CLIC4 NLS makes surprisingly favourable interactions by forming side-chain hydrogen bonds to the importin-α backbone. This structural evidence supports the hypothesis that CLIC4 translocation to the nucleus is governed by the importin-α nuclear import pathway, provided that CLIC4 can undergo a conformational rearrangement that exposes the NLS in an extended conformation., (© 2011 The Authors Journal compilation © 2011 FEBS.)
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- 2011
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25. Crystal structure of an integron gene cassette-associated protein from Vibrio cholerae identifies a cationic drug-binding module.
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Deshpande CN, Harrop SJ, Boucher Y, Hassan KA, Di Leo R, Xu X, Cui H, Savchenko A, Chang C, Labbate M, Paulsen IT, Stokes HW, Curmi PM, and Mabbutt BC
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- Amino Acid Sequence, Bacterial Proteins genetics, Cations, Conserved Sequence genetics, Crystallography, X-Ray, Ligands, Molecular Sequence Data, Phylogeny, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Sequence Alignment, Structural Homology, Protein, Bacterial Proteins chemistry, Genes, Bacterial genetics, Integrons genetics, Pharmaceutical Preparations metabolism, Vibrio cholerae genetics
- Abstract
Background: The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes., Methodology/principal Findings: We report the 1.8 Å crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators., Conclusions/significance: Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.
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- 2011
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26. The enigma of the CLIC proteins: Ion channels, redox proteins, enzymes, scaffolding proteins?
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Littler DR, Harrop SJ, Goodchild SC, Phang JM, Mynott AV, Jiang L, Valenzuela SM, Mazzanti M, Brown LJ, Breit SN, and Curmi PM
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- Animals, Cell Membrane metabolism, Chloride Channels chemistry, Chloride Channels classification, Chloride Channels genetics, Cytoskeleton metabolism, Enzymes chemistry, Enzymes classification, Enzymes genetics, Humans, Hydrogen-Ion Concentration, Oxidation-Reduction, Chloride Channels metabolism, Enzymes metabolism
- Abstract
Chloride intracellular channel proteins (CLICs) are distinct from most ion channels in that they have both soluble and integral membrane forms. CLICs are highly conserved in chordates, with six vertebrate paralogues. CLIC-like proteins are found in other metazoans. CLICs form channels in artificial bilayers in a process favoured by oxidising conditions and low pH. They are structurally plastic, with CLIC1 adopting two distinct soluble conformations. Phylogenetic and structural data indicate that CLICs are likely to have enzymatic function. The physiological role of CLICs appears to be maintenance of intracellular membranes, which is associated with tubulogenesis but may involve other substructures., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)
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- 2010
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27. Arabidopsis AtSerpin1, crystal structure and in vivo interaction with its target protease RESPONSIVE TO DESICCATION-21 (RD21).
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Lampl N, Budai-Hadrian O, Davydov O, Joss TV, Harrop SJ, Curmi PM, Roberts TH, and Fluhr R
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- Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins metabolism, Crystallography, X-Ray, Cysteine Proteases genetics, Cysteine Proteases metabolism, Mutation, Peptide Hydrolases genetics, Peptide Hydrolases metabolism, Plants, Genetically Modified, Protein Structure, Quaternary, Seedlings chemistry, Seedlings genetics, Seedlings metabolism, Serpins genetics, Serpins metabolism, Arabidopsis chemistry, Arabidopsis Proteins chemistry, Cysteine Proteases chemistry, Peptide Hydrolases chemistry, Serpins chemistry
- Abstract
In animals, protease inhibitors of the serpin family are associated with many physiological processes, including blood coagulation and innate immunity. Serpins feature a reactive center loop (RCL), which displays a protease target sequence as a bait. RCL cleavage results in an irreversible, covalent serpin-protease complex. AtSerpin1 is an Arabidopsis protease inhibitor that is expressed ubiquitously throughout the plant. The x-ray crystal structure of recombinant AtSerpin1 in its native stressed conformation was determined at 2.2 A. The electrostatic surface potential below the RCL was found to be highly positive, whereas the breach region critical for RCL insertion is an unusually open structure. AtSerpin1 accumulates in plants as a full-length and a cleaved form. Fractionation of seedling extracts by nonreducing SDS-PAGE revealed the presence of an additional slower migrating complex that was absent when leaves were treated with the specific cysteine protease inhibitor L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane. Significantly, RESPONSIVE TO DESICCATION-21 (RD21) was the major protease labeled with the L-trans-epoxysuccinyl-L-leucylamido (4-guanidino)butane derivative DCG-04 in wild type extracts but not in extracts of mutant plants constitutively overexpressing AtSerpin1, indicating competition. Fractionation by nonreducing SDS-PAGE followed by immunoblotting with RD21-specific antibody revealed that the protease accumulated both as a free enzyme and in a complex with AtSerpin1. Importantly, both RD21 and AtSerpin1 knock-out mutants lacked the serpin-protease complex. The results establish that the major Arabidopsis plant serpin interacts with RD21. This is the first report of the structure and in vivo interaction of a plant serpin with its target protease.
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- 2010
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28. Crystal structure of Lsm3 octamer from Saccharomyces cerevisiae: implications for Lsm ring organisation and recruitment.
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Naidoo N, Harrop SJ, Sobti M, Haynes PA, Szymczyna BR, Williamson JR, Curmi PM, and Mabbutt BC
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- Amino Acid Sequence, Crystallography, X-Ray, Dimerization, Magnetic Resonance Spectroscopy, Models, Biological, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, RNA, Fungal metabolism, RNA-Binding Proteins genetics, RNA-Binding Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Sequence Homology, Amino Acid, RNA-Binding Proteins chemistry, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins chemistry
- Abstract
Sm and Sm-like (Lsm) proteins are core components of the ribonucleoprotein complexes essential to key nucleic acid processing events within the eukaryotic cell. They assemble as polyprotein ring scaffolds that have the capacity to bind RNA substrates and other necessary protein factors. The crystal structure of yeast Lsm3 reveals a new organisation of the L/Sm beta-propeller ring, containing eight protein subunits. Little distortion of the characteristic L/Sm fold is required to form the octamer, indicating that the eukaryotic Lsm ring may be more pliable than previously thought. The homomeric Lsm3 octamer is found to successfully recruit Lsm6, Lsm2 and Lsm5 directly from yeast lysate. Our crystal structure shows the C-terminal tail of each Lsm3 subunit to be engaged in connections across rings through specific beta-sheet interactions with elongated loops protruding from neighbouring octamers. While these loops are of distinct length for each Lsm protein and generally comprise low-complexity polar sequences, several Lsm C-termini comprise hydrophobic sequences suitable for beta-sheet interactions. The Lsm3 structure thus provides evidence for protein-protein interactions likely utilised by the highly variable Lsm loops and termini in the recruitment of RNA processing factors to mixed Lsm ring scaffolds. Our coordinates also provide updated homology models for the active Lsm[1-7] and Lsm[2-8] heptameric rings.
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- 2008
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29. Comparison of vertebrate and invertebrate CLIC proteins: the crystal structures of Caenorhabditis elegans EXC-4 and Drosophila melanogaster DmCLIC.
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Littler DR, Harrop SJ, Brown LJ, Pankhurst GJ, Mynott AV, Luciani P, Mandyam RA, Mazzanti M, Tanda S, Berryman MA, Breit SN, and Curmi PM
- Subjects
- Animals, Binding Sites, Cations, Divalent, Crystallography, X-Ray, Drosophila melanogaster chemistry, Glutathione, Lipid Bilayers, Metals, Protein Structure, Tertiary, Caenorhabditis elegans Proteins chemistry, Chloride Channels chemistry, Drosophila Proteins chemistry
- Abstract
The crystal structures of two CLIC family members DmCLIC and EXC-4 from the invertebrates Drosophila melanogaster and Caenorhabditis elegans, respectively, have been determined. The proteins adopt a glutathione S-transferase (GST) fold. The structures are highly homologous to each other and more closely related to the known structures of the human CLIC1 and CLIC4 than to GSTs. The invertebrate CLICs show several unique features including an elongated C-terminal extension and a divalent metal binding site. The latter appears to alter the ancestral glutathione binding site, and thus, the invertebrate CLICs are unlikely to bind glutathione in the same manner as the GST proteins. Purified recombinant DmCLIC and EXC-4 both bind to lipid bilayers and can form ion channels in artificial lipid bilayers, albeit at low pH. EXC-4 differs from other CLIC proteins in that the conserved redox-active cysteine at the N-terminus of helix 1 is replaced by an aspartic acid residue. Other key distinguishing features of EXC-4 include the fact that it binds to artificial bilayers at neutral pH and this binding is not sensitive to oxidation. These differences with other CLIC family members are likely to be due to the substitution of the conserved cysteine by aspartic acid., ((c) 2007 Wiley-Liss, Inc.)
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- 2008
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30. Structural genomics of the bacterial mobile metagenome: an overview.
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Robinson A, Guilfoyle AP, Sureshan V, Howell M, Harrop SJ, Boucher Y, Stokes HW, Curmi PM, and Mabbutt BC
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins isolation & purification, Cloning, Molecular, Crystallography, X-Ray, Open Reading Frames genetics, Protein Folding, Vibrio chemistry, Bacterial Proteins chemistry, Escherichia coli genetics, Genome, Bacterial genetics, Genomics methods, Integrons physiology, Vibrio genetics
- Abstract
Mobile gene cassettes collectively carry a highly diverse pool of novel genes, ostensibly for purposes of microbial adaptation. At the sequence level, putative functions can only be assigned to a minority of carried ORFs due to their inherent novelty. Having established these mobilized genes code for folded and functional proteins, the authors have recently adopted the procedures of structural genomics to efficiently sample their structures, thereby scoping their functional range. This chapter outlines protocols used to produce cassette-associated genes as recombinant proteins in Escherichia coli and crystallization procedures based on the dual screen/pH optimization approach of the SECSG (SouthEast Collaboratory for Structural Genomics). Crystal structures solved to date have defined unique members of enzyme fold classes associated with transport and nucleotide metabolism.
- Published
- 2008
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31. A putative house-cleaning enzyme encoded within an integron array: 1.8 A crystal structure defines a new MazG subtype.
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Robinson A, Guilfoyle AP, Harrop SJ, Boucher Y, Stokes HW, Curmi PM, and Mabbutt BC
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Crystallography, X-Ray, Models, Genetic, Models, Molecular, Molecular Sequence Data, Molecular Structure, Protein Structure, Secondary, Protein Structure, Tertiary, Pyrophosphatases metabolism, Sequence Homology, Amino Acid, Substrate Specificity, Vibrio genetics, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Integrons genetics, Vibrio enzymology
- Abstract
Mobile gene cassettes collectively contain a highly diverse pool of novel genes that encode many novel adaptive functions. In the non-clinical context, the function of almost all of the encoded proteins remains unknown despite the enormous size of this mobile gene pool. We have been characterizing cassette arrays by taking advantage of the fact that they cluster at discrete sites in chromosomes; even large arrays are thus recoverable in a relatively small number of clones in genomic libraries. In one assembled array of 116 cassettes from the marine bacterium Vibrio sp. DAT722, a putative MazG protein is encoded within the 21st cassette. Because MazG proteins are implicated in a number of cellular processes, including house-cleaning and stress survival, the presence of such a protein in a mobile cassette was noteworthy. Here we solve the crystal structure of this alpha-helical protein, and define both open and closed states of a new variant of the MazG family. Functional assays confirm that the protein is a dNTP pyrophosphohydrolase, with marked preferences for dCTP and dATP. We hypothesize that iMazG acts as a house-cleaning enzyme, preventing the incorporation of damaging non-canonical nucleotides into host-cell DNA.
- Published
- 2007
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32. Plasminogen activator inhibitor-2 is highly tolerant to P8 residue substitution--implications for serpin mechanistic model and prediction of nsSNP activities.
- Author
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Di Giusto DA, Sutherland AP, Jankova L, Harrop SJ, Curmi PM, and King GC
- Subjects
- Amino Acid Substitution, Binding Sites, Crystallography, X-Ray, Enzyme Inhibitors chemistry, Humans, Kinetics, Peptides chemistry, Plasminogen Activator Inhibitor 2 physiology, Serpins genetics, Structure-Activity Relationship, Urokinase-Type Plasminogen Activator antagonists & inhibitors, Plasminogen Activator Inhibitor 2 chemistry, Plasminogen Activator Inhibitor 2 genetics, Polymorphism, Single Nucleotide, Serpins chemistry
- Abstract
The serine protease inhibitor (serpin) superfamily is involved in a wide range of cellular processes including fibrinolysis, angiogenesis, apoptosis, inflammation, metastasis and viral pathogenesis. Here, we investigate the unique mousetrap inhibition mechanism of serpins through saturation mutagenesis of the P8 residue for a typical family member, plasminogen activator inhibitor-2 (PAI-2). A number of studies have proposed an important role for the P8 residue in the efficient insertion and stabilisation of the cleaved reactive centre loop (RCL), which is a key event in the serpin inhibitory mechanism. The importance of this residue for inhibition of the PAI-2 protease target urinary plasminogen activator (urokinase, uPA) is confirmed, although a high degree of tolerance to P8 substitution is observed. Out of 19 possible PAI-2 P8 mutants, 16 display inhibitory activities within an order of magnitude of the wild-type P8 Thr species. Crystal structures of complexes between PAI-2 and RCL-mimicking peptides with P8 Met or Asp mutations are determined, and structural comparison with the wild-type complex substantiates the ability of the S8 pocket to accommodate disparate side-chains. These data indicate that the identity of the P8 residue is not a determinant of efficient RCL insertion, and provide further evidence for functional plasticity of key residues within enzyme structures. Poor correlation of observed PAI-2 P8 mutant activities with a range of physicochemical, evolutionary and thermodynamic predictive indices highlights the practical limitations of existing approaches to predicting the molecular phenotype of protein variants.
- Published
- 2005
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33. Crystal structure of the soluble form of the redox-regulated chloride ion channel protein CLIC4.
- Author
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Littler DR, Assaad NN, Harrop SJ, Brown LJ, Pankhurst GJ, Luciani P, Aguilar MI, Mazzanti M, Berryman MA, Breit SN, and Curmi PM
- Subjects
- Amino Acid Sequence, Chlorides metabolism, Crystallography, X-Ray, Electrophysiology, Humans, Liposomes metabolism, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Patch-Clamp Techniques, Protein Structure, Tertiary, Sequence Alignment, Solubility, Structural Homology, Protein, Chloride Channels chemistry, Chloride Channels metabolism
- Abstract
The structure of CLIC4, a member of the CLIC family of putative intracellular chloride ion channel proteins, has been determined at 1.8 Angstroms resolution by X-ray crystallography. The protein is monomeric and it is structurally similar to CLIC1, belonging to the GST fold class. Differences between the structures of CLIC1 and CLIC4 are localized to helix 2 in the glutaredoxin-like N-terminal domain, which has previously been shown to undergo a dramatic structural change in CLIC1 upon oxidation. The structural differences in this region correlate with the sequence differences, where the CLIC1 sequence appears to be atypical of the family. Purified, recombinant, wild-type CLIC4 is shown to bind to artificial lipid bilayers, induce a chloride efflux current when associated with artificial liposomes and produce an ion channel in artificial bilayers with a conductance of 30 pS. Membrane binding is enhanced by oxidation of CLIC4 while no channels were observed via tip-dip electrophysiology in the presence of a reducing agent. Thus, recombinant CLIC4 appears to be able to form a redox-regulated ion channel in the absence of any partner proteins.
- Published
- 2005
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34. Integron-associated mobile gene cassettes code for folded proteins: the structure of Bal32a, a new member of the adaptable alpha+beta barrel family.
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Robinson A, Wu PS, Harrop SJ, Schaeffer PM, Dosztányi Z, Gillings MR, Holmes AJ, Nevalainen KM, Stokes HW, Otting G, Dixon NE, Curmi PM, and Mabbutt BC
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Catalytic Domain, Crystallography, X-Ray, Epoxide Hydrolases chemistry, Ion Transport, Isomerases chemistry, Models, Molecular, Molecular Sequence Data, Protein Folding, Sequence Homology, Amino Acid, Soil Microbiology, Bacterial Proteins chemistry, DNA Transposable Elements, Integrons physiology, Protein Structure, Secondary, Soil analysis
- Abstract
The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.
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- 2005
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35. Developing a structure-function model for the cryptophyte phycoerythrin 545 using ultrahigh resolution crystallography and ultrafast laser spectroscopy.
- Author
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Doust AB, Marai CN, Harrop SJ, Wilk KE, Curmi PM, and Scholes GD
- Subjects
- Computer Simulation, Cryptophyta metabolism, Crystallography, X-Ray, Lasers, Models, Molecular, Photosynthesis, Phycoerythrin metabolism, Protein Conformation, Spectrophotometry, Spectrum Analysis, Cryptophyta chemistry, Phycoerythrin chemistry
- Abstract
Cryptophyte algae differ from cyanobacteria and red algae in the architecture of their photosynthetic light harvesting systems, even though all three are evolutionarily related. Central to cryptophyte light harvesting is the soluble antenna protein phycoerythrin 545 (PE545). The ultrahigh resolution crystal structure of PE545, isolated from a unicellular cryptophyte Rhodomonas CS24, is reported at both 1.1A and 0.97A resolution, revealing details of the conformation and environments of the chromophores. Absorption, emission and polarized steady state spectroscopy (298K, 77K), as well as ultrafast (20fs time resolution) measurements of population dynamics are reported. Coupled with complementary quantum chemical calculations of electronic transitions of the bilins, these enable assignment of spectral absorption characteristics to each chromophore in the structure. Spectral differences between the tetrapyrrole pigments due to chemical differences between bilins, as well as their binding and interaction with the local protein environment are described. Based on these assignments, and considering customized optical properties such as strong coupling, a model for light harvesting by PE545 is developed which explains the fast, directional harvesting of excitation energy. The excitation energy is funnelled from four peripheral pigments (beta158,beta82) into a central chromophore dimer (beta50/beta61) in approximately 1ps. Those chromophores, in turn, transfer the excitation energy to the red absorbing molecules located at the periphery of the complex in approximately 4ps. A final resonance energy transfer step sensitizes just one of the alpha19 bilins on a time scale of 22ps. Furthermore, it is concluded that binding of PE545 to the thylakoid membrane is not essential for efficient energy transfer to the integral membrane chlorophyll a-containing complexes associated with PS-II.
- Published
- 2004
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36. The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition.
- Author
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Littler DR, Harrop SJ, Fairlie WD, Brown LJ, Pankhurst GJ, Pankhurst S, DeMaere MZ, Campbell TJ, Bauskin AR, Tonini R, Mazzanti M, Breit SN, and Curmi PM
- Subjects
- Amino Acid Sequence, Chloride Channels metabolism, Dimerization, Electrophysiology, Humans, Molecular Sequence Data, Oxidation-Reduction, Protein Conformation, Sequence Alignment, Structure-Activity Relationship, Chloride Channels chemistry
- Abstract
Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.
- Published
- 2004
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37. Homomeric ring assemblies of eukaryotic Sm proteins have affinity for both RNA and DNA. Crystal structure of an oligomeric complex of yeast SmF.
- Author
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Collins BM, Cubeddu L, Naidoo N, Harrop SJ, Kornfeld GD, Dawes IW, Curmi PM, and Mabbutt BC
- Subjects
- Binding Sites, DNA chemistry, DNA metabolism, Dimerization, Models, Molecular, Protein Binding, RNA chemistry, RNA metabolism, Ribonucleoproteins, Small Nuclear metabolism, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae Proteins metabolism, Ribonucleoproteins, Small Nuclear chemistry, Saccharomyces cerevisiae Proteins chemistry
- Abstract
Sm and Sm-like proteins are key components of small ribonucleoproteins involved in many RNA and DNA processing pathways. In eukaryotes, these complexes contain seven unique Sm or Sm-like (Lsm) proteins assembled as hetero-heptameric rings, whereas in Archaea and bacteria six or seven-membered rings are made from only a single polypeptide chain. Here we show that single Sm and Lsm proteins from yeast also have the capacity to assemble into homo-oligomeric rings. Formation of homo-oligomers by the spliceosomal small nuclear ribonucleoprotein components SmE and SmF preclude hetero-interactions vital to formation of functional small nuclear RNP complexes in vivo. To better understand these unusual complexes, we have determined the crystal structure of the homomeric assembly of the spliceosomal protein SmF. Like its archaeal/bacterial homologs, the SmF complex forms a homomeric ring but in an entirely novel arrangement whereby two heptameric rings form a co-axially stacked dimer via interactions mediated by the variable loops of the individual SmF protein chains. Furthermore, we demonstrate that the homomeric assemblies of yeast Sm and Lsm proteins are capable of binding not only to oligo(U) RNA but, in the case of SmF, also to oligo(dT) single-stranded DNA.
- Published
- 2003
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38. Recombinant CLIC1 (NCC27) assembles in lipid bilayers via a pH-dependent two-state process to form chloride ion channels with identical characteristics to those observed in Chinese hamster ovary cells expressing CLIC1.
- Author
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Warton K, Tonini R, Fairlie WD, Matthews JM, Valenzuela SM, Qiu MR, Wu WM, Pankhurst S, Bauskin AR, Harrop SJ, Campbell TJ, Curmi PM, Breit SN, and Mazzanti M
- Subjects
- Animals, CHO Cells, Chloride Channels genetics, Chlorides metabolism, Circular Dichroism, Cricetinae, Electrophysiology, Hydrogen-Ion Concentration, Kinetics, Liposomes, Protein Conformation, Recombinant Proteins metabolism, Spectrometry, Fluorescence, Chloride Channels metabolism, Lipid Bilayers metabolism
- Abstract
CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.
- Published
- 2002
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39. Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution.
- Author
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Harrop SJ, DeMaere MZ, Fairlie WD, Reztsova T, Valenzuela SM, Mazzanti M, Tonini R, Qiu MR, Jankova L, Warton K, Bauskin AR, Wu WM, Pankhurst S, Campbell TJ, Breit SN, and Curmi PM
- Subjects
- Amino Acid Sequence, Binding Sites, Cell Membrane metabolism, Chlorine metabolism, Cysteine chemistry, Electrophysiology, Escherichia coli metabolism, Glutathione metabolism, Glutathione Transferase metabolism, Humans, Lipid Bilayers metabolism, Models, Molecular, Molecular Sequence Data, Oxidation-Reduction, Patch-Clamp Techniques, Point Mutation, Protein Binding, Protein Conformation, Protein Structure, Tertiary, Recombinant Fusion Proteins metabolism, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Chloride Channels chemistry, Chlorine chemistry
- Abstract
CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.
- Published
- 2001
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40. Interaction between the P14 residue and strand 2 of beta-sheet B is critical for reactive center loop insertion in plasminogen activator inhibitor-2.
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Saunders DN, Jankova L, Harrop SJ, Curmi PM, Gould AR, Ranson M, and Baker MS
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- Amino Acids chemistry, Electrophoresis, Polyacrylamide Gel, Humans, Hydrogen Bonding, Models, Biological, Models, Chemical, Models, Molecular, Mutation, Peptides chemistry, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins pharmacology, Spectrometry, Fluorescence, Threonine chemistry, Urea pharmacology, Plasminogen Activator Inhibitor 2 chemistry, Plasminogen Activator Inhibitor 2 metabolism
- Abstract
The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.
- Published
- 2001
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41. Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop.
- Author
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Jankova L, Harrop SJ, Saunders DN, Andrews JL, Bertram KC, Gould AR, Baker MS, and Curmi PM
- Subjects
- Electrons, Escherichia coli metabolism, Gene Deletion, Glycine chemistry, Humans, Models, Molecular, Mutation, Protein Binding, Protein Conformation, Protein Structure, Secondary, Protein Structure, Tertiary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Serpins chemistry, Threonine chemistry, Crystallography, X-Ray, Peptides chemistry, Plasminogen Activator Inhibitor 2 chemistry
- Abstract
The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.
- Published
- 2001
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42. Crystal structure of a heptameric Sm-like protein complex from archaea: implications for the structure and evolution of snRNPs.
- Author
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Collins BM, Harrop SJ, Kornfeld GD, Dawes IW, Curmi PM, and Mabbutt BC
- Subjects
- Amino Acid Sequence, Archaeal Proteins genetics, Archaeal Proteins metabolism, Binding Sites, Conserved Sequence, Crystallography, X-Ray, Gene Order, Hydrogen Bonding, Methanobacterium genetics, Models, Molecular, Molecular Sequence Data, Protein Structure, Quaternary, Protein Structure, Tertiary, Protein Subunits, RNA genetics, RNA metabolism, Ribonucleoproteins, Small Nuclear genetics, Ribonucleoproteins, Small Nuclear metabolism, Ribosomal Proteins genetics, Sequence Alignment, Archaeal Proteins chemistry, Evolution, Molecular, Methanobacterium chemistry, Ribonucleoproteins, Small Nuclear chemistry
- Abstract
The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsm alpha protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A resolution. The Lsm alpha protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via beta-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel beta-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsm alpha is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsm alpha complex in ribosome function or biogenesis., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
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43. Evolution of a light-harvesting protein by addition of new subunits and rearrangement of conserved elements: crystal structure of a cryptophyte phycoerythrin at 1.63-A resolution.
- Author
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Wilk KE, Harrop SJ, Jankova L, Edler D, Keenan G, Sharples F, Hiller RG, and Curmi PM
- Subjects
- Computer Graphics, Conserved Sequence, Crystallography, X-Ray methods, Dimerization, Energy Transfer, Macromolecular Substances, Models, Molecular, Molecular Sequence Data, Phycobilisomes, Protein Conformation, Protein Structure, Secondary, Eukaryota metabolism, Phycoerythrin chemistry
- Abstract
Cryptophytes are unicellular photosynthetic algae that use a lumenally located light-harvesting system, which is distinct from the phycobilisome structure found in cyanobacteria and red algae. One of the key components of this system is water-soluble phycoerythrin (PE) 545 whose expression is enhanced by low light levels. The crystal structure of the heterodimeric alpha(1)alpha(2)betabeta PE 545 from the marine cryptophyte Rhodomonas CS24 has been determined at 1.63-A resolution. Although the beta-chain structure is similar to the alpha and beta chains of other known phycobiliproteins, the overall structure of PE 545 is novel with the alpha chains forming a simple extended fold with an antiparallel beta-ribbon followed by an alpha-helix. The two doubly linked beta50/beta61 chromophores (one on each beta subunit) are in van der Waals contact, suggesting that exciton-coupling mechanisms may alter their spectral properties. Each alpha subunit carries a covalently linked 15,16-dihydrobiliverdin chromophore that is likely to be the final energy acceptor. The architecture of the heterodimer suggests that PE 545 may dock to an acceptor protein via a deep cleft and that energy may be transferred via this intermediary protein to the reaction center.
- Published
- 1999
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44. Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion.
- Author
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Hädener A, Matzinger PK, Battersby AR, McSweeney S, Thompson AW, Hammersley AP, Harrop SJ, Cassetta A, Deacon A, Hunter WN, Nieh YP, Raftery J, Hunter N, and Helliwell JR
- Subjects
- Binding Sites, Crystallography, X-Ray, Data Collection, Models, Molecular, Protein Conformation, Recombinant Proteins chemistry, Selenium chemistry, Temperature, Hydroxymethylbilane Synthase chemistry, Selenomethionine chemistry
- Abstract
The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.
- Published
- 1999
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45. The crystal structure of plasminogen activator inhibitor 2 at 2.0 A resolution: implications for serpin function.
- Author
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Harrop SJ, Jankova L, Coles M, Jardine D, Whittaker JS, Gould AR, Meister A, King GC, Mabbutt BC, and Curmi PM
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, Humans, Models, Molecular, Molecular Sequence Data, Protein Folding, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Alignment, Sequence Deletion, Sequence Homology, Amino Acid, Plasminogen Activator Inhibitor 2 chemistry, Plasminogen Activator Inhibitor 2 metabolism, Serpins chemistry, Serpins metabolism
- Abstract
Background: Plasminogen activator inhibitor 2 (PAI-2) is a member of the serpin family of protease inhibitors that function via a dramatic structural change from a native, stressed state to a relaxed form. This transition is mediated by a segment of the serpin termed the reactive centre loop (RCL); the RCL is cleaved on interaction with the protease and becomes inserted into betasheet A of the serpin. Major questions remain as to what factors facilitate this transition and how they relate to protease inhibition., Results: The crystal structure of a mutant form of human PAI-2 in the stressed state has been determined at 2.0 A resolution. The RCL is completely disordered in the structure. An examination of polar residues that are highly conserved across all serpins identifies functionally important regions. A buried polar cluster beneath betasheet A (the so-called 'shutter' region) is found to stabilise both the stressed and relaxed forms via a rearrangement of hydrogen bonds., Conclusions: A statistical analysis of interstrand interactions indicated that the shutter region can be used to discriminate between inhibitory and non-inhibitory serpins. This analysis implied that insertion of the RCL into betasheet A up to residue P8 is important for protease inhibition and hence the structure of the complex formed between the serpin and the target protease.
- Published
- 1999
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46. Structure of a human lysosomal sulfatase.
- Author
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Bond CS, Clements PR, Ashby SJ, Collyer CA, Harrop SJ, Hopwood JJ, and Guss JM
- Subjects
- Alkaline Phosphatase chemistry, Amino Acid Sequence, Animals, Binding Sites, CHO Cells, Chondro-4-Sulfatase antagonists & inhibitors, Chondro-4-Sulfatase deficiency, Chondro-4-Sulfatase genetics, Consensus Sequence, Cricetinae, Crystallography, X-Ray, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Glycosylation, Humans, Models, Molecular, Molecular Sequence Data, Mucopolysaccharidosis VI enzymology, Mucopolysaccharidosis VI genetics, Multigene Family, Point Mutation, Recombinant Proteins antagonists & inhibitors, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Sequence Alignment, Sequence Homology, Amino Acid, Vanadates metabolism, Vanadates pharmacology, Chondro-4-Sulfatase chemistry, Lysosomes enzymology, Protein Conformation
- Abstract
Background: . Sulfatases catalyze the hydrolysis of sulfuric acid esters from a wide variety of substrates including glycosaminoglycans, glycolipids and steroids. There is sufficient common sequence similarity within the class of sulfatase enzymes to indicate that they have a common structure. Deficiencies of specific lysosomal sulfatases that are involved in the degradation of glycosamino-glycans lead to rare inherited clinical disorders termed mucopolysaccharidoses. In sufferers of multiple sulfatase deficiency, all sulfatases are inactive because an essential post-translational modification of a specific active-site cysteine residue to oxo-alanine does not occur. Studies of this disorder have contributed to location and characterization of the sulfatase active site. To understand the catalytic mechanism of sulfatases, and ultimately the determinants of their substrate specificities, we have determined the structure of N-acetylgalactosamine-4-sulfatase., Results: . The crystal structure of the enzyme has been solved and refined at 2.5 resolution using data recorded at both 123K and 273K. The structure has two domains, the larger of which belongs to the alpha/beta class of proteins and contains the active site. The enzyme active site in the crystals contains several hitherto undescribed features. The active-site cysteine residue, Cys91, is found as the sulfate derivative of the aldehyde species, oxo-alanine. The sulfate is bound to a previously undetected metal ion, which we have identified as calcium. The structure of a vanadate-inhibited form of the enzyme has also been solved, and this structure shows that vanadate has replaced sulfate in the active site and that the vanadate is covalently linked to the protein. Preliminary data is presented for crystals soaked in the monosaccharide N-acetylgalactosamine, the structure of which forms a product complex of the enzyme., Conclusions: . The structure of N-acetylgalactosamine-4-sulfatase reveals that residues conserved amongst the sulfatase family are involved in stabilizing the calcium ion and the sulfate ester in the active site. This suggests an archetypal fold for the family of sulfatases. A catalytic role is proposed for the post-translationally modified highly conserved cysteine residue. Despite a lack of any previously detectable sequence similarity to any protein of known structure, the large sulfatase domain that contains the active site closely resembles that of alkaline phosphatase: the calcium ion in sulfatase superposes on one of the zinc ions in alkaline phosphatase and the sulfate ester of Cys91 superposes on the phosphate ion found in the active site of alkaline phosphatase.
- Published
- 1997
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47. Trends and challenges in experimental macromolecular crystallography.
- Author
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Chayen NE, Boggon TJ, Cassetta A, Deacon A, Gleichmann T, Habash J, Harrop SJ, Helliwell JR, Nieh YP, Peterson MR, Raftery J, Snell EH, Hädener A, Niemann AC, Siddons DP, Stojanoff V, Thompson AW, Ursby T, and Wulff M
- Subjects
- Biophysics trends, Concanavalin A chemistry, Concanavalin A isolation & purification, Crystallization, Crystallography, X-Ray, Electrochemistry, Interferometry, Light, Macromolecular Substances, Microscopy, Atomic Force, Microscopy, Video, Neutrons, Scattering, Radiation, Solubility, Synchrotrons, Weightlessness, Crystallography methods, Proteins chemistry, Proteins isolation & purification
- Published
- 1996
- Full Text
- View/download PDF
48. Structure solution of a cubic crystal of concanavalin A complexed with methyl alpha-D-glucopyranoside.
- Author
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Harrop SJ, Helliwell JR, Wan TC, Kalb AJ, Tong L, and Yariv J
- Abstract
The solution of the cubic crystal form (a = 167.8 A) of concanavalin A complexed with the monosaccharide methyl alpha-D-glucopyranoside is described. The space group has been determined as I2(1)3 rather than I23. The use of cadmium to replace cobalt at the transition metal-ion binding site and to replace calcium at its binding site proved to be crucial to the successful solution of the crystal structure. The relatively small isomorphous signals of 21 e(-) for the replacement of cobalt and 28 e(-) for the replacement of calcium, yielded interpretable difference Patterson maps. The electron-density map calculated in space group I2(1)3 at 5.4 A resolution, based on phases derived from single- and double-substituted cadmium differences, revealed a classical concanavalin A tetramer of 222 point symmetry, as seen in all the known crystal structures of concanavalin A. Rigid-body refinement at 3.6 A using the refined coordinates of saccharide-free concanavalin A converged to an R factor of 27.4%. A molecular-replacement analysis, consistent with this crystal structure, and initial experiences in the incorrect space group I23 are described as these also prove to be instructive.
- Published
- 1996
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49. MAD Phasing Strategies Explored with a Brominated Oligonucleotide Crystal at 1.65A Resolution.
- Author
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Peterson MR, Harrop SJ, McSweeney SM, Leonard GA, Thompson AW, Hunter WN, and Helliwell JR
- Abstract
The crystal structure of a brominated oligonucleotide d(CGCG(Br)CG), chemical formula C(114)N(48)O(68)P(10)Br(2), has been analysed by multiwavelength anomalous dispersion (MAD) methods. The oligonucleotide crystallizes in space group P2(1)2(1)2(1) with a = 17.97, b = 30.98, c = 44.85 A, alpha = beta = gamma 90 degrees . Data to a resolution of 1.65 A were collected at four wavelengths about the K-absorption edge of the bromine atom (lambda(1) = 0.9323 A, a reference wavelength at the long-wavelength side of the edge; lambda(2) = 0.9192 A, at the absorption-edge inflection point; lambda(3) = 0.9185 A, at the ;white line' absorption maximum; lambda(4) = 0.8983 A, a reference wavelength at the short-wavelength side) using synchrotron radiation at Station PX9.5, SRS, Daresbury. Multiwavelength data could be collected on a single-crystal as the sample was radiation stable. Anomalous and dispersive Patterson maps were readily interpretable to give the bromine anomalous scatterer positions. Phase calculations to 1.65 A, resolution, using all four wavelengths, gave a figure of merit of 0.825 for 2454 reflections. The electron-density map was readily interpretable showing excellent connectivity for the sugar/phosphate backbone and each base was easily characterized. The two nucleotide strands paired up as expected in an antiparallel Watson-Crick-type manner. The structure was refined to 1.65 A using all the data (R-factor = 17.0% based on 3151 reflections, with a data-to-parameter ratio of 2.6). In addition to the four-wavelength analysis, a variety of other phasing strategies, and the associated quality of the resulting electron-density maps, were compared. These included use of either of the reference wavelength data sets in the two possible three-wavelength phasing combinations to assess their relative effectiveness. Moreover, the time dependence upon measuring the Bijvoet differences and its effect upon phasing was also investigated. Finally, the use of only two wavelengths, including Friedel pairs, is demonstrated (the theoretical minimum case); this is of particular interest when considering overall beam time needs and is clearly a feasible experimental strategy, as shown here.
- Published
- 1996
- Full Text
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50. Additional crystal forms of the E. coli class II fructose-1,6-bisphosphate aldolase.
- Author
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Kitagawa Y, Leonard GA, Harrop SJ, Peterson MR, Hunter WN, Qamar S, and Berry A
- Abstract
We have obtained two additional crystal forms of the metal-dependent class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of elongated plates have unit-cell dimensions a = 73.4, b = 120.0, c = 190.1 A, orthorhombic space group P2(1)2(1)2(1). Monoclinic prisms have unit-cell dimensions a = 67.7, b = 104.3, c = 52.8 A, beta = 105 degrees, space group P2(1). Diffraction to slightly better than 3.0 A, has been observed for both forms using in-house and synchrotron facilities. These crystal forms may aid the structure solution of this enzyme by presenting additional forms for heavy-atom derivatization. These forms have multiple copies of the enzyme in the asymmetric unit and averaging methods might also be useful in the analysis.
- Published
- 1995
- Full Text
- View/download PDF
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