18 results on '"Proteau, Ariane"'
Search Results
2. Structure of [NiFe] hydrogenase maturation protein HypE from Escherichia coli and its interaction with HypF
- Author
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Rangarajan, Erumbi S., Asinas, Abdalin, Proteau, Ariane, Munger, Christine, Baardsnes, Jason, Iannuzzi, Pietro, Matte, Allan, and Cygler, Miroslaw
- Subjects
Escherichia coli -- Physiological aspects ,Bacterial proteins -- Structure ,Bacterial proteins -- Properties ,Hydrogenation -- Research ,Crystals -- Structure ,Crystals -- Research ,Biological sciences - Abstract
Hydrogenases are enzymes involved in hydrogen metabolism, utilizing [H.sub.2] as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-[Angstrom] resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a [K.sub.d] (dissociation constant) of ~400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to forma stoichiometric, hetero-oligomeric complex predominantly consisting of a [[EF].sub.2] heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction.
- Published
- 2008
3. Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7†
- Author
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Rangarajan, Erumbi S., Ruane, Karen M., Proteau, Ariane, Schrag, Joseph D., Valladares, Ricardo, Gonzalez, Claudio F., Gilbert, Michel, Yakunin, Alexander F., and Cygler, Miroslaw
- Published
- 2011
- Full Text
- View/download PDF
4. Structure and active site residues of PdID, an N-acetyltransferase from the bacillosamine synthetic pathway required for N-glycan synthesis in Campylobacter jejuni
- Author
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Rangarajan, Erumbi S., Ruane, Karen M., Sulea, Traian, Watson, David C., Proteau, Ariane, Leclerc, Sonia, Cygler, Miroslaw, Matte, Allan, and Young, N. Martin
- Subjects
Campylobacter -- Composition ,Glycoproteins -- Structure ,Glycoproteins -- Chemical properties ,Protein folding -- Research ,Biological sciences ,Chemistry - Abstract
A study was conducted to determine the crystal structure and function of PgID enzyme in CoA-bound and unbound forms isolated from Campylobacter jejuni, highly unusual bacteria forming N-linked glycoproteins. The enzyme is found to be the member of L[beta]H family of complex sugar N-acetyltransferases which adopts an extended structure in the unbound form and folds to extend the [beta]-helix upon binding CoA.
- Published
- 2008
5. Structural and functional analysis of Campylobacter jejuni PseG : A UDP-sugar hydrolase from the pseudaminic acid biosynthetic pathway
- Author
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Rangarajan, Erumbi S., Proteau, Ariane, Cui, Qizhi, Logan, Susan M., Potetinova, Zhanna, Whitfield, Dennis, Purisima, Enrico O., Cygler, Miroslaw, Matte, Allan, Sulea, Traian, and Schoenhofen, Ian C.
- Abstract
Flagella of the bacteria Helicobacter pylori and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido- 2,4,6-trideoxy-β-L-altropyranose to generate 2,4-diacetamido- 2,4,6-trideoxy-L-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His¹⁷-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His¹⁷ functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C–O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family.
- Published
- 2009
6. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.
- Author
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D’Costa, Vanessa M., Braun, Virginie, Landekic, Marija, Shi, Rong, Proteau, Ariane, McDonald, Laura, Cygler, Miroslaw, Grinstein, Sergio, and Brumell, John H.
- Abstract
Summary Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
7. Structural Basis for Fe-S Cluster Assembly and tRNA Thiolation Mediated by IscS Protein-Protein Interactions.
- Author
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Rong Shi, Proteau, Ariane, Villarroya, Magda, Moukadiri, Ismaïl, Linhua Zhang, Trempe, Jean-François, Matte, Allan, Armengod, M. Eugenia, and Cygler, Miroslaw
- Subjects
- *
CYSTEINE desulfurase , *ENZYMES , *PHYSIOLOGICAL effects of sulfur , *BIOSYNTHESIS , *URIDINE , *PROTEINS - Abstract
The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters. [ABSTRACT FROM AUTHOR]
- Published
- 2010
- Full Text
- View/download PDF
8. Structural and Functional Analysis of Campylobacter jejuni PseG.
- Author
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Rangarajan, Erumbi S., Proteau, Ariane, Qizhi Cui, Logan, Susan M., Potetinova, Zhanna, Whitfield, Dennis, Purisima, Enrico O., CygIer, Miroslaw, Matte, Allan, Sulea, Traian, and Schoenhofen, Ian C.
- Subjects
- *
FLAGELLA (Microbiology) , *HELICOBACTER pylori , *CAMPYLOBACTER jejuni , *GLYCOSYLATION , *SIALIC acids , *HYDROLYSIS , *HYDROLASES - Abstract
Flagella of the bacteria Helicobacter pylon and Campylobacter jejuni are important virulence determinants, whose proper assembly and function are dependent upon glycosylation at multiple positions by sialic acid-like sugars, such as 5,7diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid (Pse)). The fourth enzymatic step in the pseudaminic acid pathway, the hydrolysis of UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose to generate 2,4-diacetamido-2,4,6-trideoxy-L-altropyranose, is performed by the nucleotide sugar hydrolase PseG. To better understand the molecular basis of the PseG catalytic reaction, we have determined the crystal structures of C. jejuni PseG in apo-form and as a complex with its UDP product at 1.8 and 1.85 Å resolution, respectively. In addition, molecular modeling was utilized to provide insight into the structure of the PseG-substrate complex. This modeling identifies a His17-coordinated water molecule as the putative nucleophile and suggests the UDP-sugar substrate adopts a twist-boat conformation upon binding to PseG, enhancing the exposure of the anomeric bond cleaved and favoring inversion at C-1. Furthermore, based on these structures a series of amino acid substitution derivatives were constructed, altering residues within the active site, and each was kinetically characterized to examine its contribution to PseG catalysis. In conjunction with structural comparisons, the almost complete inactivation of the PseG H17F and H17L derivatives suggests that His17 functions as an active site base, thereby activating the nucleophilic water molecule for attack of the anomeric C-O bond of the UDP-sugar. As the PseG structure reveals similarity to those of glycosyltransferase family-28 members, in particular that of Escherichia coli MurG, these findings may also be of relevance for the mechanistic understanding of this important enzyme family. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
9. Trapping open and closed forms of FitE-A group III periplasmic binding protein.
- Author
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Shi, Rong, Proteau, Ariane, Wagner, John, Cui, Qizhi, Purisima, Enrico O., Matte, Allan, and Cygler, Miroslaw
- Abstract
Periplasmic binding proteins (PBPs) are essential components of bacterial transport systems, necessary for bacterial growth and survival. The two-domain structures of PBPs are topologically classified into three groups based on the number of crossovers or hinges between the globular domains: group I PBPs have three connections, group II have two, and group III have only one. Although a large number of structures for group I or II PBPs are known, fewer group III PBPs have been structurally characterized. Group I and II PBPs exhibit significant domain motions during transition from the unbound to ligand-bound form, however, no large conformational changes have been observed to date in group III PBPs. We have solved the crystal structure of a periplasmic binding protein FitE, part of an iron transport system, fit, recently identified in a clinical E. coli isolate. The structure, determined at 1.8 Å resolution, shows that FitE is a group III PBP containing a single α-helix bridging the two domains. Among the individual FitE molecules present in two crystal forms we observed three different conformations (open, closed, intermediate). Our crystallographic and molecular dynamics results strongly support the notion that group III PBPs also adopt the same Venus flytrap mechanism as do groups I and II PBPs. Unlike other group III PBPs, FitE forms dimers both in solution and in the crystals. The putative siderophore binding pocket is lined with arginine residues, suggesting an anionic nature of the iron-containing siderophore. Proteins 2009. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
10. Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.
- Author
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Tocilj, Ante, Munger, Christine, Proteau, Ariane, Morona, Renato, Purins, Leanne, Ajamian, Eunice, Wagner, John, Papadopoulos, Magdalene, Van Den Bosch, Luisa, Rubinstein, John L., Féthière, James, Matte, Allan, and Cygler, Miroslaw
- Subjects
ENZYMES ,POLYSACCHARIDES ,BACTERIA ,POLYMERS ,ELECTRON microscopy ,OLIGOMERS - Abstract
The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central α-helix extending 100 Å into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery. [ABSTRACT FROM AUTHOR]
- Published
- 2008
- Full Text
- View/download PDF
11. Structural Snapshots of Escherichia coli Histidinol Phosphate Phosphatase along the Reaction Pathway.
- Author
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Rangarajan, Erumbi S., Proteau, Ariane, Wagner, John, Ming-Ni Hung, Matte, Allan, and Cygler, Miroslaw
- Subjects
- *
ENZYMES , *BIOSYNTHESIS , *HALOACID dehalogenase , *CATALYSIS , *MOLECULAR structure , *DNA polymerases , *RNA polymerases , *BIOCHEMISTRY - Abstract
HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of L-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn2+ ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg2+, Mn2+, Co2+, or Zn2+, but Ca2+ has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg2+ ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca2+. [ABSTRACT FROM AUTHOR]
- Published
- 2006
- Full Text
- View/download PDF
12. The Multifunctional Nuclear Protein p54nrb is Multiphosphorylated in Mitosis and Interacts with the Mitotic Regulator Pin1
- Author
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Proteau, Ariane, Blier, Stéphanie, Albert, Alexandra L., Lavoie, Sébastien B., Traish, Abdulmaged M., and Vincent, Michel
- Subjects
- *
PROTEINS , *MITOSIS , *CELL division , *NUCLEIC acids - Abstract
The human protein p54nrb and its mouse homolog NonO have been implicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA relaxation. We have identified p54nrb as an antigen of the phosphodependent monoclonal antibodies CC-3 and MPM-2 and shown that this protein is phosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletion studies with an anti-cyclin B1 antibody established that Cdk1 was responsible for the phosphorylation of the carboxy-terminal extremity of p54nrb whereas a different kinase appeared to be involved in the generation of CC-3 epitope(s) in the amino-terminal moiety of the protein. Like many CC-3 and MPM-2 antigens, we show that p54nrb is a target of the peptidylprolyl isomerase Pin1, suggesting that it may be regulated by phosphorylation-dependent conformational changes as many other nuclear proteins upon entry into mitosis. In addition, site-directed mutagenesis indicated that the interaction of Pin1 with p54nrb was mediated by three threonine residues located in the proline-rich carboxy-terminal extremity of the protein. Our results also showed that Pin1 binding was favored when at least two of the three threonine residues were phosphorylated, suggesting a regulation mechanism based on multisite phosphorylation. [Copyright &y& Elsevier]
- Published
- 2005
- Full Text
- View/download PDF
13. Structure and Function of the Glycopeptide N-methyltransferase MtfA, a Tool for the Biosynthesis of Modified Glycopeptide Antibiotics
- Author
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Shi, Rong, Lamb, Sherry S., Zakeri, Bijan, Proteau, Ariane, Cui, Qizhi, Sulea, Traian, Matte, Allan, Wright, Gerard D., and Cygler, Miroslaw
- Subjects
- *
DRUGS , *SULFAGUANIDINE , *ANTIBIOTICS , *ANTISEPTICS - Abstract
Summary: There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs. [Copyright &y& Elsevier]
- Published
- 2009
- Full Text
- View/download PDF
14. Structure-Function Analysis of Escherichia coli MnmG (GidA), a Highly Conserved tRNA-Modifying Enzyme.
- Author
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Rong Shi, Villarroya, Magda, Ruiz-Partida, Rafael, Yunge Li, Proteau, Ariane, Prado, Silvia, Moukadiri, Ismaïl, Benítez-Páez, Alfonso, Lomas, Rodrigo, Wagner, John, Matte, Allan, Velázquez-Campoy, Adrián, Armengod, M.-Eugenia, and Cygler, Miroslaw
- Subjects
- *
ESCHERICHIA coli , *TRANSFER RNA , *ESCHERICHIA , *ENZYMES , *ENTEROBACTERIACEAE , *FLAVINS , *ADENINE nucleotides , *GENETIC mutation , *GENETICS - Abstract
The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-Å resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
15. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7.
- Author
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D'Costa VM, Braun V, Landekic M, Shi R, Proteau A, McDonald L, Cygler M, Grinstein S, and Brumell JH
- Subjects
- Adaptor Proteins, Signal Transducing metabolism, Amino Acid Sequence, Animals, Bacterial Proteins chemistry, DNA-Binding Proteins metabolism, Endosomes microbiology, HEK293 Cells, HeLa Cells, Humans, Mice, Microtubule-Associated Proteins, Molecular Sequence Data, Protein Binding, Protein Structure, Tertiary, Protein Transport, Salmonella enterica metabolism, Salmonella enterica pathogenicity, Transcription Factors metabolism, rab7 GTP-Binding Proteins, Bacterial Proteins metabolism, Endosomes metabolism, rab GTP-Binding Proteins metabolism
- Abstract
Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
16. Application of dynamic light scattering in protein crystallization.
- Author
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Proteau A, Shi R, and Cygler M
- Subjects
- Crystallography, X-Ray, Light, Scattering, Radiation, Crystallization methods, Proteins chemistry
- Abstract
Success in determining the three-dimensional structure of a macromolecule by X-ray diffraction methods depends critically on the ability to obtain well ordered crystals of the macromolecule in question. Predisposition to crystallization correlates with the homogeneity of the molecules in solution. Dynamic light scattering (DLS) is particularly well suited for evaluating protein homogeneity under multiple conditions and at concentrations commensurate with crystallization conditions. This unit presents a typical protocol for DLS measurements of a protein sample, and describes approaches to improve protein homogeneity in solution.
- Published
- 2010
- Full Text
- View/download PDF
17. Structural basis for Fe-S cluster assembly and tRNA thiolation mediated by IscS protein-protein interactions.
- Author
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Shi R, Proteau A, Villarroya M, Moukadiri I, Zhang L, Trempe JF, Matte A, Armengod ME, and Cygler M
- Subjects
- Carbon-Sulfur Lyases chemistry, Carbon-Sulfur Lyases genetics, Catalytic Domain, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Iron-Sulfur Proteins genetics, Iron-Sulfur Proteins metabolism, Models, Molecular, Molecular Sequence Data, Protein Binding, RNA, Transfer metabolism, Sulfur chemistry, Sulfur metabolism, Carbon-Sulfur Lyases metabolism, Escherichia coli Proteins chemistry, Iron-Sulfur Proteins chemistry, Protein Conformation, RNA, Transfer chemistry, Sulfhydryl Compounds chemistry
- Abstract
The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters., Competing Interests: The authors have declared that no competing interests exist.
- Published
- 2010
- Full Text
- View/download PDF
18. The multifunctional nuclear protein p54nrb is multiphosphorylated in mitosis and interacts with the mitotic regulator Pin1.
- Author
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Proteau A, Blier S, Albert AL, Lavoie SB, Traish AM, and Vincent M
- Subjects
- Animals, CDC2 Protein Kinase metabolism, Epitopes immunology, HeLa Cells, Humans, Mice, NIMA-Interacting Peptidylprolyl Isomerase, Nuclear Matrix-Associated Proteins genetics, Nuclear Proteins genetics, Phosphorylation, Protein Binding, Protein Serine-Threonine Kinases immunology, Protein Serine-Threonine Kinases metabolism, RNA-Binding Proteins genetics, Substrate Specificity, Mitosis, Nuclear Matrix-Associated Proteins metabolism, Nuclear Proteins metabolism, Peptidylprolyl Isomerase metabolism, RNA-Binding Proteins metabolism
- Abstract
The human protein p54nrb and its mouse homolog NonO have been implicated in a variety of nuclear processes including transcription, pre-mRNA processing, nuclear retention of edited RNA and DNA relaxation. We have identified p54nrb as an antigen of the phosphodependent monoclonal antibodies CC-3 and MPM-2 and shown that this protein is phosphorylated on multiple sites during mitosis. The use of the cyclin-dependent protein kinase inhibitor roscovitine and immunodepletion studies with an anti-cyclin B1 antibody established that Cdk1 was responsible for the phosphorylation of the carboxy-terminal extremity of p54nrb whereas a different kinase appeared to be involved in the generation of CC-3 epitope(s) in the amino-terminal moiety of the protein. Like many CC-3 and MPM-2 antigens, we show that p54nrb is a target of the peptidylprolyl isomerase Pin1, suggesting that it may be regulated by phosphorylation-dependent conformational changes as many other nuclear proteins upon entry into mitosis. In addition, site-directed mutagenesis indicated that the interaction of Pin1 with p54nrb was mediated by three threonine residues located in the proline-rich carboxy-terminal extremity of the protein. Our results also showed that Pin1 binding was favored when at least two of the three threonine residues were phosphorylated, suggesting a regulation mechanism based on multisite phosphorylation.
- Published
- 2005
- Full Text
- View/download PDF
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