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1.
PLoS Pathog ; 18(7): e1010664, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35793393

RESUMO

Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans, causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses Rlm3- and Rlm9-mediated resistance triggered by AvrLm3 and AvrLm5-9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous AVR effector candidate from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the AVR effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues are named LARS (for Leptosphaeria AviRulence and Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered oilseed rape immunity in several genotypes carrying Rlm3. Furthermore, this resistance could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 shares a common activity with AvrLm3 within the host plant which is detected by Rlm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3.


Assuntos
Brassica napus , Doenças das Plantas , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Virulência/genética
2.
Microb Cell Fact ; 23(1): 70, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38419080

RESUMO

We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.108 cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.


Assuntos
Magnetossomos , Magnetospirillum , Magnetospirillum/genética , Ferro , Preparações Farmacêuticas , Proteínas de Bactérias/genética
3.
Plant J ; 83(4): 610-24, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26082394

RESUMO

The avirulence gene AvrLm4-7 of Leptosphaeria maculans, the causal agent of stem canker in Brassica napus (oilseed rape), confers a dual specificity of recognition by two resistance genes (Rlm4 and Rlm7) and is strongly involved in fungal fitness. In order to elucidate the biological function of AvrLm4-7 and understand the specificity of recognition by Rlm4 and Rlm7, the AvrLm4-7 protein was produced in Pichia pastoris and its crystal structure was determined. It revealed the presence of four disulfide bridges, but no close structural analogs could be identified. A short stretch of amino acids in the C terminus of the protein, (R/N)(Y/F)(R/S)E(F/W), was well-conserved among AvrLm4-7 homologs. Loss of recognition of AvrLm4-7 by Rlm4 is caused by the mutation of a single glycine to an arginine residue located in a loop of the protein. Loss of recognition by Rlm7 is governed by more complex mutational patterns, including gene loss or drastic modifications of the protein structure. Three point mutations altered residues in the well-conserved C-terminal motif or close to the glycine involved in Rlm4-mediated recognition, resulting in the loss of Rlm7-mediated recognition. Transient expression in Nicotiana benthamiana (tobacco) and particle bombardment experiments on leaves from oilseed rape suggested that AvrLm4-7 interacts with its cognate R proteins inside the plant cell, and can be translocated into plant cells in the absence of the pathogen. Translocation of AvrLm4-7 into oilseed rape leaves is likely to require the (R/N)(Y/F)(R/S)E(F/W) motif as well as an RAWG motif located in a nearby loop that together form a positively charged region.


Assuntos
Ascomicetos/patogenicidade , Brassica napus/metabolismo , Brassica napus/microbiologia , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Virulência/genética
4.
Protein Expr Purif ; 114: 121-7, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26145834

RESUMO

The eukaryotic lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KA in higher eukaryotes) is a ubiquitous enzyme that synthesizes the plasma membrane pool of phosphatidylinositol 4-phosphate. This important phosphoinositide has key roles in different signalization pathways, vesicular traffic and cellular compartment identity. Moreover, human PI4K4A is an essential factor for hepatitis C virus replication. PI4KA is a large protein (2102 residues for human PI4KA) with the kinase domain making up the ca 400 C-terminal residues. There is essentially no structural information about the 1500N-terminal residues and no clue as to the function of most of this region of PI4KA. In this report, we use computational methods in order to delineate fragments of human PI4KA amenable to soluble production in Escherichia coli. We clone and express these fragments as GST-fusions and evaluate the soluble fraction of each protein. Finally, we produce and purify to homogeneity a 1100-residue PI4KA N-terminal fragment. Our results further suggest that PI4KA can be described as a two-module protein. They open the way to structural characterization of the N-terminal regulatory module of PI4KA.


Assuntos
1-Fosfatidilinositol 4-Quinase/isolamento & purificação , 1-Fosfatidilinositol 4-Quinase/metabolismo , Escherichia coli/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , 1-Fosfatidilinositol 4-Quinase/química , 1-Fosfatidilinositol 4-Quinase/genética , Sequência de Aminoácidos , Biologia Computacional , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência
5.
Structure ; 16(1): 52-61, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18184583

RESUMO

Loss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNA(Phe) has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.


Assuntos
RNA Fúngico/química , RNA de Transferência de Fenilalanina/química , Saccharomyces cerevisiae/química , Sítios de Ligação , Cristalografia por Raios X , Guanosina/análogos & derivados , Modelos Moleculares , Conformação de Ácido Nucleico , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , RNA de Transferência de Fenilalanina/genética , RNA de Transferência de Fenilalanina/isolamento & purificação , Saccharomyces cerevisiae/genética , Difração de Raios X
6.
Artigo em Inglês | MEDLINE | ID: mdl-32133346

RESUMO

We report the synthesis in large quantity of highly pure magnetosomes for medical applications. For that, magnetosomes are produced by MSR-1 Magnetospirillum gryphiswaldense magnetotactic bacteria using minimal growth media devoid of uncharacterized and toxic products prohibited by pharmaceutical regulation, i.e., yeast extract, heavy metals different from iron, and carcinogenic, mutagenic and reprotoxic agents. This method follows two steps, during which bacteria are first pre-amplified without producing magnetosomes and are then fed with an iron source to synthesize magnetosomes, yielding, after 50 h of growth, an equivalent OD565 of ~8 and 10 mg of magnetosomes in iron per liter of growth media. Compared with magnetosomes produced in non-minimal growth media, those particles have lower concentrations in metals other than iron. Very significant reduction or disappearance in magnetosome composition of zinc, manganese, barium, and aluminum are observed. This new synthesis method paves the way towards the production of magnetosomes for medical applications.

7.
Methods Mol Biol ; 363: 21-37, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17272835

RESUMO

The South-Paris Yeast Structural Genomics Pilot Project (http://www.genomics.eu.org) aims at systematically expressing, purifying, and determining the three-dimensional structures of Saccharomyces cerevisiae proteins. We have already cloned 240 yeast open reading frames in the Escherichia coli pET system. Eighty-two percent of the targets can be expressed in E. coli, and 61% yield soluble protein. We have currently purified 58 proteins. Twelve X-ray structures have been solved, six are in progress, and six other proteins gave crystals. In this chapter, we present the general experimental flowchart applied for this project. One of the main difficulties encountered in this pilot project was the low solubility of a great number of target proteins. We have developed parallel strategies to recover these proteins from inclusion bodies, including refolding, coexpression with chaperones, and an in vitro expression system. A limited proteolysis protocol, developed to localize flexible regions in proteins that could hinder crystallization, is also described.


Assuntos
Proteínas/metabolismo , Sequência de Aminoácidos , Clonagem Molecular , Biologia Computacional , Cristalização , Genômica , Peptídeo Hidrolases/metabolismo , Proteínas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Protein Sci ; 15(6): 1516-21, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16731983

RESUMO

The Saccharomyces cerevisiae His6 gene codes for the enzyme phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase, catalyzing the fourth step in histidine biosynthesis. To get an insight into the structure and function of this enzyme, we determined its X-ray structure at a resolution of 1.30 A using the anomalous diffraction signal of the protein's sulphur atoms at 1.77 A wavelength. His6 folds in an (alpha/beta)8 barrel similar to HisA, which performs the same function in bacteria and archaea. We found a citrate molecule from the buffer bound in a pocket near the expected position of the active site and used it to model the open form of the substrate (phosphoribulosyl moiety), which is a reaction intermediate. This model enables us to identify catalytic residues and to propose a reaction mechanism where two aspartates act as acid/base catalysts: Asp134 as a proton donor for ring opening, and Asp9 as a proton acceptor and donor during enolization of the aminoaldose. Asp9 is conserved in yeast His6 and bacterial or archaeal HisA sequences, and Asp134 has equivalents in both HisA and TrpF, but they occur at a different position in the protein sequence.


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Aminoidrolases/química , Aminoidrolases/metabolismo , Ácido Aspártico/metabolismo , Sítios de Ligação , Domínio Catalítico , Citratos/metabolismo , Cristalografia por Raios X/métodos , Modelos Moleculares , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Enxofre/química
9.
J Mol Biol ; 345(5): 1131-9, 2005 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-15644210

RESUMO

Annexin A8 is a relatively infrequent and poorly studied member of this large family of calcium-binding and membrane-binding proteins. It is, however, associated with a specific disease, acute promyelocytic leukemia. We have solved its three-dimensional structure, which includes a moderately long and intact N terminus. The structure is closest to that of annexin A3 and highlights several important regions of inherent flexibility in the annexin molecule. The N terminus resembles that of annexin A3, as it lies along the concave surface of the molecule and inserts partially into the hydrophilic channel in its centre. Since both annexins A3 and A8 are expressed in promyelocytic cells during their differentiation, the similarity in their structures might suggest a functional relationship.


Assuntos
Anexina A3/química , Anexinas/química , Anexinas/genética , Anexinas/metabolismo , Sítios de Ligação , Cálcio/farmacologia , Cristalografia por Raios X , Lisina/genética , Lisina/metabolismo , Modelos Moleculares , Mutação/genética , Conformação Proteica
10.
Protein Sci ; 14(10): 2751-8, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16195557

RESUMO

We present here the structure of Yer010c protein of unknown function, solved by Multiple Anomalous Diffraction and revealing a common fold and oligomerization state with proteins of the regulator of ribonuclease activity A (RraA) family. In Escherichia coli, RraA has been shown to regulate the activity of ribonuclease E by direct interaction. The absence of ribonuclease E in yeast suggests a different function for this family member in this organism. Yer010cp has a few supplementary secondary structure elements and a deep pseudo-knot at the heart of the protein core. A tunnel at the interface between two monomers, lined with conserved charged residues, has unassigned residual electron density and may constitute an active site for a yet unknown activity.


Assuntos
Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Cristalografia por Raios X , Motivos Nó de Cisteína , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Família Multigênica/genética , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
11.
Protein Sci ; 14(1): 209-15, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15608122

RESUMO

We determined the three-dimensional crystal structure of the protein YML079wp, encoded by a hypothetical open reading frame from Saccharomyces cerevisiae to a resolution of 1.75 A. The protein has no close homologs and its molecular and cellular functions are unknown. The structure of the protein is a jelly-roll fold consisting of ten beta-strands organized in two parallel packed beta-sheets. The protein has strong structural resemblance to the plant storage and ligand binding proteins (canavalin, glycinin, auxin binding protein) but also to some plant and bacterial enzymes (epimerase, germin). The protein forms homodimers in the crystal, confirming measurements of its molecular mass in solution. Two monomers have their beta-sheet packed together to form the dimer. The presence of a hydrophobic ligand in a well conserved pocket inside the barrel and local sequence similarity with bacterial epimerases may suggest a biochemical function for this protein.


Assuntos
Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
12.
Proteins ; 60(4): 778-86, 2005 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-16021630

RESUMO

In the Pseudomonas bacterial genomes, the PhzF proteins are involved in the production of phenazine derivative antibiotic and antifungal compounds. The PhzF superfamily however also encompasses proteins in all genomes from bacteria to eukaryotes, for which no function has been assigned. We have determined the three dimensional crystal structure at 2.05 A resolution of YHI9, the yeast member of the PhzF family. YHI9 has a fold similar to bacterial diaminopimelate epimerase, revealing a bimodular structure with an internal symmetry. Residue conservation identifies a putative active site at the interface between the two domains. Evolution of this protein by gene duplication, gene fusion and domain swapping from an ancestral gene containing the "hot dog" fold, identifies the protein as a "kinked double hot dog" fold.


Assuntos
Isomerases de Aminoácido/química , Proteínas de Saccharomyces cerevisiae/química , Isomerases de Aminoácido/genética , Isomerases de Aminoácido/isolamento & purificação , Cristalografia por Raios X , Modelos Moleculares , Reação em Cadeia da Polimerase , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação
13.
Proteins ; 54(4): 776-83, 2004 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-14997573

RESUMO

The protein product of the YGR205w gene of Saccharomyces cerevisiae was targeted as part of our yeast structural genomics project. YGR205w codes for a small (290 amino acids) protein with unknown structure and function. The only recognizable sequence feature is the presence of a Walker A motif (P loop) indicating a possible nucleotide binding/converting function. We determined the three-dimensional crystal structure of Se-methionine substituted protein using multiple anomalous diffraction. The structure revealed a well known mononucleotide fold and strong resemblance to the structure of small metabolite phosphorylating enzymes such as pantothenate and phosphoribulo kinase. Biochemical experiments show that YGR205w binds specifically ATP and, less tightly, ADP. The structure also revealed the presence of two bound sulphate ions, occupying opposite niches in a canyon that corresponds to the active site of the protein. One sulphate is bound to the P-loop in a position that corresponds to the position of beta-phosphate in mononucleotide protein ATP complex, suggesting the protein is indeed a kinase. The nature of the phosphate accepting substrate remains to be determined.


Assuntos
Escherichia coli/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato , Sulfatos/metabolismo
14.
Artigo em Inglês | MEDLINE | ID: mdl-15236693

RESUMO

The aim of the present work was to develop a highly productive and simplified process for active human galectin-1 (Gal1) production. Gal1 is a beta-galactoside binding lectin that differentially affects biological and cellular functions such as immune surveillance and apoptosis. These effects have attracted the attention of researchers in cell biology, biochemistry and immunology. However, the production of sufficient amounts of recombinant human Gal1 (rhGal1) is needed to study of the effects of Gal1 during cell treatments. To this end, an high-yield expression of rhGal1 was achieved by high-cell density fed-batch cultivation using an exponential glycerol feeding strategy and rhGal1 was purified by a one-step purification scheme using affinity chromatography.


Assuntos
Galectina 1/isolamento & purificação , Escherichia coli/genética , Galectina 1/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
15.
Mol Biotechnol ; 56(9): 839-48, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24859267

RESUMO

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50-65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100% of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.


Assuntos
6-Fitase/biossíntese , 6-Fitase/genética , Bacillus subtilis/enzimologia , Pichia/genética , 6-Fitase/química , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Clonagem Molecular , Estabilidade Enzimática , Glicosilação , Pichia/metabolismo , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
16.
FEBS J ; 281(24): 5513-31, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25290767

RESUMO

Streptococcus equi is a horse pathogen belonging to Lancefield group C. Infection by S. equi ssp. equi causes strangles, a serious and highly contagious disease of the upper respiratory tract. S. equi ssp. equi secretes a fibronectin (Fn)-binding protein, FNE, that does not contain cell wall-anchoring motifs. FNE binds to the gelatin-binding domain (GBD) of Fn, composed of the motifs (6) FI (12) FII (789) FI . FNE lacks the canonical Fn-binding peptide repeats observed in many microbial surface components recognizing adhesive matrix molecules. We found that the interaction between FNE and the human GBD is mediated by the binding of the disordered C-terminal region (residues 208-262) of FNE to the (789) FI GBD subfragment. The crystal structure of FNE showed that it is similar to the minor pilus protein Spy0125 of Streptococcus pyogenes, found at the end of pilus polymers and responsible for adhesion. FNE and Spy0125 both have a superimposable internal thioester bond between highly conserved Cys and Gln residues. Small-angle X-ray scattering of the FNE-(789) FI complex provided a model that aligns the C-terminal peptide of FNE with the E-strands of the FI domains, adopting the ß-zipper extension model observed in previous structures of microbial surface components recognizing adhesive matrix molecule adhesion peptides bound to FI domains.


Assuntos
Adesinas Bacterianas/metabolismo , Streptococcus equi/metabolismo , Adesinas Bacterianas/química , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Dados de Sequência Molecular , Conformação Proteica , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos
17.
J Mol Biol ; 398(5): 641-6, 2010 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-20359485

RESUMO

Flavin adenine dinucleotide (FAD) synthetase is an essential enzyme responsible for the synthesis of FAD by adenylation of riboflavin monophosphate (FMN). We have solved the 1.9 A resolution structure of Fad1, the yeast FAD synthetase, in complex with the FAD product in the active site. The structure of Fad1 shows it to be a member of the PP-ATPase superfamily. Important conformational differences in the two motifs involved in binding the phosphate moieties of FAD compared to the Candida glabrata FMNT ortholog suggests that this loop is dynamic and undergoes substantial conformational changes during its catalytic cycle.


Assuntos
Flavina-Adenina Dinucleotídeo/química , Nucleotidiltransferases/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Candida glabrata/química , Candida glabrata/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Flavina-Adenina Dinucleotídeo/metabolismo , Modelos Moleculares , Nucleotidiltransferases/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
18.
Protein Sci ; 17(8): 1336-45, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18511537

RESUMO

The genome of Pyrococcus abyssi contains two open reading frames encoding proteins which had been previously predicted to be DNA ligases, Pab2002 and Pab1020. We show that while the former is indeed a DNA ligase, Pab1020 had no effect on the substrate deoxyoligo-ribonucleotides tested. Instead, Pab1020 catalyzes the nucleotidylation of oligo-ribonucleotides in an ATP-dependent reaction, suggesting that it is an RNA ligase. We have solved the structure of Pab1020 in complex with the ATP analog AMPPNP by single-wavelength anomalous dispersion (SAD), elucidating a structure with high structural similarity to the catalytic domains of two RNA ligases from the bacteriophage T4. Additional carboxy-terminal domains are also present, and one of these mediates contacts with a second protomer, which is related by noncrystallographic symmetry, generating a homodimeric structure. These C-terminal domains are terminated by short domain swaps which themselves end within 5 A of the active sites of the partner molecules. Additionally, we show that the protein is indeed capable of circularizing RNA molecules in an ATP-dependent reaction. These structural and biochemical results provide an insight into the potential physiological roles of Pab1020.


Assuntos
Proteínas Arqueais/química , DNA Ligases/química , Pyrococcus abyssi/enzimologia , RNA Ligase (ATP)/química , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bacteriófago T4/enzimologia , Domínio Catalítico , Cristalografia por Raios X , DNA Ligase Dependente de ATP , DNA Ligases/genética , DNA Ligases/metabolismo , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pyrococcus abyssi/genética , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , Homologia de Sequência de Aminoácidos , Proteínas Virais/química , Proteínas Virais/metabolismo
19.
Extremophiles ; 11(2): 403-13, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17091222

RESUMO

We present here the experimental strategies, first results and identified bottlenecks of a structural genomics initiative on membrane proteins of the hyperthermophilic archaea Pyrococcus abyssi. Five ORFs coding for putative membrane proteins have been cloned and expressed in the methylotrophic Pichia pastoris expression system, using two different constructs, with or without the signal sequence alpha-mating factor of Saccharomyces cerevisiae. A c-myc epitope and 6 His codons were added at the 3'-end of the targeted genes to allow immunodetection of the recombinant proteins and to facilitate their further purification. We have selected at least one producer clone for each protein of interest and for almost every construction. All the membrane proteins were produced in Erlenmeyer flasks culture and in fed-batch cultivation for large-scale preparation. The proteins were detected in the membrane fractions of P. pastoris. Production efficiencies were relatively low in both production conditions but the quantities of biomass obtained during fed-batch cultivation have allowed us to collect sufficient amount of material for further purification. The proteins were extracted, solubilized and partially purified. Large-scale purification will be necessary for further structural work.


Assuntos
Proteínas Arqueais/biossíntese , Expressão Gênica , Proteínas de Membrana/biossíntese , Pichia/crescimento & desenvolvimento , Pyrococcus abyssi/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Arqueais/genética , Proteínas de Membrana/genética , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética
20.
Acta Crystallogr D Biol Crystallogr ; 58(Pt 7): 1243-5, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12077457

RESUMO

The Escherichia coli protein YodA was overexpressed, purified and crystallized in several crystal forms. The function of this protein is not known, although it has been identified under conditions of bacterial stress. Three of the four crystal forms were obtained in the presence of divalent cations (zinc, nickel and cadmium), suggesting that YodA may be a metal-binding protein.


Assuntos
Cristalografia por Raios X/métodos , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Cádmio/química , Níquel/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Zinco/química
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