RESUMO
We have determined the crystal structure of DR1281 from Deinococcus radiodurans. DR1281 is a protein of unknown function with over 170 homologs found in prokaryotes and eukaryotes. To elucidate the molecular function of DR1281, its crystal structure at 2.3 A resolution was determined and a series of biochemical screens for catalytic activity was performed. The crystal structure shows that DR1281 has two domains, a small alpha domain and a putative catalytic domain formed by a four-layered structure of two beta-sheets flanked by five alpha-helices on both sides. The small alpha domain interacts with other molecules in the asymmetric unit and contributes to the formation of oligomers. The structural comparison of the putative catalytic domain with known structures suggested its biochemical function to be a phosphatase, phosphodiesterase, nuclease, or nucleotidase. Structural analyses with its homologues also indicated that there is a dinuclear center at the interface of two domains formed by Asp8, Glu37, Asn38, Asn65, His148, His173, and His175. An absolute requirement of metal ions for activity has been proved by enzymatic assay with various divalent metal ions. A panel of general enzymatic assays of DR1281 revealed metal-dependent catalytic activity toward model substrates for phosphatases (p-nitrophenyl phosphate) and phosphodiesterases (bis-p-nitrophenyl phosphate). Subsequent secondary enzymatic screens with natural substrates demonstrated significant phosphatase activity toward phosphoenolpyruvate and phosphodiesterase activity toward 2',3'-cAMP. Thus, our structural and enzymatic studies have identified the biochemical function of DR1281 as a novel phosphatase/phosphodiesterase and disclosed key conserved residues involved in metal binding and catalytic activity.
Assuntos
Proteínas de Bactérias/química , Calcineurina/química , Deinococcus/enzimologia , Diester Fosfórico Hidrolases/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Calcineurina/metabolismo , Dados de Sequência Molecular , Diester Fosfórico Hidrolases/metabolismo , Conformação Proteica , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
DnaA is an essential component in the initiation of bacterial chromosomal replication. DnaA binds to a series of 9 base pair repeats leading to oligomerization, recruitment of the DnaBC helicase, and the assembly of the replication fork machinery. The structure of the N-terminal domain (residues 1-100) of DnaA from Mycoplasma genitalium was determined by NMR spectroscopy. The backbone r.m.s.d. for the first 86 residues was 0.6 +/- 0.2 A based on 742 NOE, 50 hydrogen bond, 46 backbone angle, and 88 residual dipolar coupling restraints. Ultracentrifugation studies revealed that the domain is monomeric in solution. Features on the protein surface include a hydrophobic cleft flanked by several negative residues on one side, and positive residues on the other. A negatively charged ridge is present on the opposite face of the protein. These surfaces may be important sites of interaction with other proteins involved in the replication process. Together, the structure and NMR assignments should facilitate the design of new experiments to probe the protein-protein interactions essential for the initiation of DNA replication.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Espectroscopia de Ressonância Magnética/métodos , Mycoplasma genitalium/metabolismo , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Replicação do DNA , DnaB Helicases/metabolismo , Escherichia coli/metabolismo , Ligação de Hidrogênio , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade da EspécieAssuntos
Bacillus/química , Bacillus/genética , Proteínas de Bactérias/química , Transativadores/química , Sequência de Aminoácidos , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Cristalografia por Raios X , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Transativadores/biossíntese , Transativadores/genéticaAssuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Mycoplasma pneumoniae/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
We have determined the crystal structure of the DUF16 domain of unknown function encoded by the gene MPN010 of Mycoplasma pneumoniae at 1.8 A resolution. The crystal structure revealed that this domain is composed of two separated homotrimeric coiled-coils. The shorter one consists of 11 highly conserved residues. The sequence comprises noncanonical heptad repeats that induce a right-handed coiled-coil structure. The longer one is composed of approximately nine heptad repeats. In this coiled-coil structure, there are three distinguishable regions that confer unique structural properties compared with other known homotrimeric coiled-coils. The first part, containing one stutter, is an unusual phenylalanine-rich region that is not found in any other coiled-coil structures. The second part is a highly conserved glutamine-rich region, frequently found in other trimeric coiled-coil structures. The last part is composed of prototype heptad repeats. The phylogenetic analysis of the DUF16 family together with a secondary structure prediction shows that the DUF16 family can be classified into five subclasses according to N-terminal sequences. Based on the structural comparison with other coiled-coil structures, a probable molecular function of the DUF16 family is discussed.
Assuntos
Mycoplasma pneumoniae/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Dados de Sequência Molecular , Mycoplasma pneumoniae/enzimologia , Filogenia , Dobramento de Proteína , Estrutura Secundária de Proteína , Alinhamento de Sequência , Análise de SequênciaRESUMO
Structural maintenance of chromosome (SMC) proteins are essential in chromosome condensation and interact with non-SMC proteins in eukaryotes and with segregation and condensation proteins (ScpA and ScpB) in prokaryotes. The highly conserved gene in Chlorobium tepidum gi 21646405 encodes ScpB (ScpB_ChTe). The high resolution crystal structure of ScpB_ChTe shows that the monomeric structure consists of two similarly shaped globular domains composed of three helices sided by beta-strands [a winged helix-turn-helix (HTH)], a motif observed in the C-terminal domain of Scc1, a functionally related eukaryotic ScpA homolog, as well as in many DNA binding proteins.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Chlorobium/genética , Sequência de Aminoácidos , Cromossomos Bacterianos/genética , Cristalografia por Raios X , Dados de Sequência Molecular , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Phosphotransacetylase (Pta) [EC 2.3.1.8] plays a major role in acetate metabolism by catalyzing the reversible transfer of the acetyl group between coenzyme A (CoA) and orthophosphate: CH(3)COSCoA+HPO(4)(2-)<-->CH(3)COOPO(3)(2-) +CoASH. In this study, we report the crystal structures of Pta from Bacillus subtilis at 2.75 A resolution and its complex with acetyl phosphate, one of its substrates, at 2.85 A resolution. In addition, the Pta activity of the enzyme has been assayed. The enzyme folds into an alpha/beta architecture with two domains separated by a prominent cleft, very similar to two other known Pta structures. The enzyme-acetyl phosphate complex structure reveals a few potential substrate binding sites. Two of them are located in the middle of the interdomain cleft: each one is surrounded by a region of strictly and highly conserved residues. High structural similarities are found with 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA), and isocitrate and isopropylmalate dehydrogenases, all of which utilize NADP+ as their cofactor, which binds in the interdomain cleft. Their substrate binding sites are close to the acetyl phosphate binding sites of Pta in the cleft as well. These results suggest that the CoA is likely to bind to the interdomain cleft of Pta in a similar way as NADP+ binds to the other three enzymes.
Assuntos
Bacillus subtilis/enzimologia , Organofosfatos/química , Fosfato Acetiltransferase/química , Fosfato Acetiltransferase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Coenzima A/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Organofosfatos/metabolismo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
NAD kinase is a ubiquitous enzyme that catalyzes the phosphorylation of NAD to NADP using ATP or inorganic polyphosphate (poly(P)) as phosphate donor, and is regarded as the only enzyme responsible for the synthesis of NADP. We present here the crystal structures of an NAD kinase from the archaeal organism Archaeoglobus fulgidus in complex with its phosphate donor ATP at 1.7 A resolution, with its substrate NAD at 3.05 A resolution, and with the product NADP in two different crystal forms at 2.45 A and 2.0 A resolution, respectively. In the ATP bound structure, the AMP portion of the ATP molecule is found to use the same binding site as the nicotinamide ribose portion of NAD/NADP in the NAD/NADP bound structures. A magnesium ion is found to be coordinated to the phosphate tail of ATP as well as to a pyrophosphate group. The conserved GGDG loop forms hydrogen bonds with the pyrophosphate group in the ATP-bound structure and the 2' phosphate group of the NADP in the NADP-bound structures. A possible phosphate transfer mechanism is proposed on the basis of the structures presented.
Assuntos
Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/enzimologia , NADP/metabolismo , NAD/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of 'unique' protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated 'hypothetical proteins' inferred their molecular functions.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano/genética , Modelos Moleculares , Mycoplasma genitalium/genética , Mycoplasma pneumoniae/genética , Dobramento de Proteína , Proteômica/métodos , Clonagem Molecular , CristalizaçãoRESUMO
The crystal structure of the hypothetical protein MPN555 from Mycoplasma pneumoniae (gi|1673958) has been determined to a resolution of 2.8 Angstrom using anomalous diffraction data at the Se-peak wavelength. Structure determination revealed a mostly alpha-helical protein with a three-lobed shape. The three lobes or fingers delineate a central binding groove and additional grooves between lobes 1 and 3 and between lobes 2 and 3. For one of the molecules in the asymmetric unit, the central binding pocket was filled with a peptide from the uncleaved N-terminal affinity tag. The MPN555 structure has structural homology to two bacterial chaperone proteins: SurA and trigger factor from Escherichia coli. The structural data and the homology to other chaperone proteins suggests an involvement in protein folding as a molecular chaperone for MPN555.
Assuntos
Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Mycoplasma/metabolismo , Peptidilprolil Isomerase/química , Sequência de Aminoácidos , Proteínas de Transporte/metabolismo , Clonagem Molecular , Cristalografia por Raios X , Primers do DNA/química , Elétrons , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Chaperonas Moleculares , Dados de Sequência Molecular , Peptidilprolil Isomerase/metabolismo , Reação em Cadeia da Polimerase , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Pseudomonas/metabolismo , Ureaplasma/metabolismo , Vibrio cholerae/metabolismo , Difração de Raios XRESUMO
The crystal structure of a hypothetical protein, TM1457, from Thermotoga maritima has been determined at 2.0A resolution. TM1457 belongs to the DUF464 family (57 members) for which there is no known function. The structure shows that it is composed of two helices in contact with one side of a five-stranded beta-sheet. Two identical monomers form a pseudo-dimer in the asymmetric unit. There is a large cleft between the first alpha-helix and the second beta-strand. This cleft may be functionally important, since the two highly conserved motifs, GHA and VCAXV(S/T), are located around the cleft. A structural comparison of TM1457 with known protein structures shows the best hit with another hypothetical protein, Ybl001C from Saccharomyces cerevisiae, though they share low structural similarity. Therefore, TM1457 still retains a unique topology and reveals a novel fold.
Assuntos
Proteínas de Bactérias/química , Cristalografia por Raios X , Thermotoga maritima/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Sequência Conservada , Dimerização , Escherichia coli/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coli. Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2A, respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.
Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/química , Fosfatos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/fisiologia , Sequência Conservada , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
All cells have a defense mechanism against a sudden heat-shock stress. Commonly, they express a set of proteins that protect cellular proteins from being denatured by heat. Among them, GroE and DnaK chaperones are representative defending systems, and their transcription is regulated by a heat-shock repressor protein HrcA. HrcA repressor controls the transcription of groE and dnaK operons by binding the palindromic CIRCE element, presumably as a dimer, and the activity of HrcA repressor is modulated by GroE chaperones. Here, we report the first crystal structure of a heat-inducible transcriptional repressor, HrcA, from Thermotoga maritima at 2.2A resolution. The Tm_HrcA protein crystallizes as a dimer. The monomer is composed of three domains: an N-terminal winged helix-turn-helix domain (WH), a GAF-like domain, and an inserted dimerizing domain (IDD). The IDD shows a unique structural fold with an anti-parallel beta-sheet composed of three beta-strands sided by four alpha-helices. The Tm_HrcA dimer structure is formed through hydrophobic contact between the IDDs and a limited contact that involves conserved residues between the GAF-like domains. In the overall dimer structure, the two WH domains are exposed, but the conformation of these two domains seems to be incompatible with DNA binding. We suggest that our structure may represent an inactive form of the HrcA repressor. Structural implication on how the inactive form of HrcA may be converted to the active form by GroEL binding to a conserved C-terminal sequence region of HrcA is discussed.
Assuntos
Proteínas Repressoras/química , Thermotoga maritima/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Chaperonina 60/metabolismo , Sequência Conservada , Cristalografia por Raios X , Dimerização , Interações Hidrofóbicas e Hidrofílicas , Estrutura Molecular , Conformação Proteica , Proteínas Repressoras/metabolismoAssuntos
Proteínas Ferro-Enxofre/química , Streptococcus pyogenes/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Sequência Conservada , Cristalografia por Raios X , Primers do DNA , Proteínas Ferro-Enxofre/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão , Proteínas Recombinantes/química , Zinco/metabolismoRESUMO
We have determined the crystal structure of nicotinate phosphoribosyltransferase from Themoplasma acidophilum (TaNAPRTase). The TaNAPRTase has three domains, an N-terminal domain, a central functional domain, and a unique C-terminal domain. The crystal structure revealed that the functional domain has a type II phosphoribosyltransferase fold that may be a common architecture for both nicotinic acid and quinolinic acid (QA) phosphoribosyltransferases (PRTase) despite low sequence similarity between them. Unlike QAPRTase, TaNAPRTase has a unique extra C-terminal domain containing a zinc knuckle-like motif containing 4 cysteines. The TaNAPRTase forms a trimer of dimers in the crystal. The active site pocket is formed at dimer interfaces. The complex structures with phosphoribosylpyrophosphate (PRPP) and nicotinate mononucleotide (NAMN) showed, surprisingly, that functional residues lining on the active site of TaNAPRTase are quite different from those of QAPRTase, although their substrates are quite similar to each other. The phosphate moiety of PRPP and NAMN is anchored to the phosphate-binding loops formed by backbone amides, as found in many alpha/beta barrel enzymes. The pyrophosphate moiety of PRPP is located at the entrance of the active site pocket, whereas the nicotinate moiety of NAMN is located deep inside. Interestingly, the nicotinate moiety of NAMN is intercalated between highly conserved aromatic residues Tyr(21) and Phe(138). Careful structural analyses combined with other NAPRTase sequence subfamilies reveal that TaNAPRTase represents a unique sequence subfamily of NAPRTase. The structures of TaNAPRTase also provide valuable insight for other sequence subfamilies such as pre-B cell colony-enhancing factor, known to have nicotinamide phosphoribosyltransferase activity.
Assuntos
Proteínas Arqueais/química , Pentosiltransferases/química , Thermoplasma/enzimologia , Sequência de Aminoácidos , Proteínas Arqueais/isolamento & purificação , Sítios de Ligação , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Pentosiltransferases/isolamento & purificaçãoRESUMO
Type I restriction-modification enzymes are differentiated from type II and type III enzymes by their recognition of two specific dsDNA sequences separated by a given spacer and cleaving DNA randomly away from the recognition sites. They are oligomeric proteins formed by three subunits: a specificity subunit, a methylation subunit, and a restriction subunit. We solved the crystal structure of a specificity subunit from Methanococcus jannaschii at 2.4-A resolution. Two highly conserved regions (CRs) in the middle and at the C terminus form a coiled-coil of long antiparallel alpha-helices. Two target recognition domains form globular structures with almost identical topologies and two separate DNA binding clefts with a modeled DNA helix axis positioned across the CR helices. The structure suggests that the coiled-coil CRs act as a molecular ruler for the separation between two recognized DNA sequences. Furthermore, the relative orientation of the two DNA binding clefts suggests kinking of bound dsDNA and exposing of target adenines from the recognized DNA sequences.
Assuntos
Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Enzimas de Restrição do DNA/química , Mathanococcus/genética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
PhoU proteins are known to play a role in the regulation of phosphate uptake. In Thermotoga maritima, two PhoU homologues have been identified bioinformatically. Here we report the crystal structure of one of the PhoU homologues at 2.0 A resolution. The structure of the PhoU protein homologue contains a highly symmetric new structural fold composed of two repeats of a three-helix bundle. The structure unexpectedly revealed a trinuclear and a tetranuclear iron cluster that were found to be bound on the surface. Each of the two multinuclear iron clusters is coordinated by a conserved E(D)XXXD motif pair. Our structure reveals a new class of metalloprotein containing multinuclear iron clusters. The possible functional implication based on the structure are discussed.
Assuntos
Ferro/metabolismo , Thermotoga maritima/metabolismo , Fatores de Transcrição/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Espectrometria de Massas , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Fatores de Transcrição/genéticaRESUMO
NAD kinase is the only known enzyme that catalyzes the formation of NADP, a coenzyme involved in most anabolic reactions and in the antioxidant defense system. Despite its importance, very little is known regarding the mechanism of catalysis and only recently have several NAD kinase structures been deposited in the PDB. Here, an independent investigation of the crystal structure of inorganic polyphosphate/ATP-NAD kinase, PPNK_THEMA, a protein from Thermotoga maritima, is reported at a resolution of 2.3 A. The crystal structure was solved using single-wavelength anomalous diffraction (SAD) data collected at the Se absorption-peak wavelength in a state in which no cofactors or substrates were bound. It revealed that the 258-amino-acid protein is folded into two distinct domains, similar to recently reported NAD kinases. The N-terminal alpha/beta-domain spans the first 100 amino acids and the last 30 amino acids of the polypeptide and has several topological matches in the PDB, whereas the other domain, which spans the middle 130 residues, adopts a unique beta-sandwich architecture and only appreciably matches the recently deposited PDB structures of NAD kinases.