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1.
Palanivelu DV Goepfert A Meury M Guye P Dehio C Schirmer T 《Protein science : a publication of the Protein Society》2011,20(3):492-499
Numerous bacterial pathogens subvert cellular functions of eukaryotic host cells by the injection of effector proteins via dedicated secretion systems. The type IV secretion system (T4SS) effector protein BepA from Bartonella henselae is composed of an N‐terminal Fic domain and a C‐terminal Bartonella intracellular delivery domain, the latter being responsible for T4SS‐mediated translocation into host cells. A proteolysis resistant fragment (residues 10–302) that includes the Fic domain shows autoadenylylation activity and adenylyl transfer onto Hela cell extract proteins as demonstrated by autoradiography on incubation with α‐[32P]‐ATP. Its crystal structure, determined to 2.9‐Å resolution by the SeMet‐SAD method, exhibits the canonical Fic fold including the HPFxxGNGRxxR signature motif with several elaborations in loop regions and an additional β‐rich domain at the C‐terminus. On crystal soaking with ATP/Mg2+, additional electron density indicated the presence of a PPi/Mg2+ moiety, the side product of the adenylylation reaction, in the anion binding nest of the signature motif. On the basis of this information and that of the recent structure of IbpA(Fic2) in complex with the eukaryotic target protein Cdc42, we present a detailed model for the ternary complex of Fic with the two substrates, ATP/Mg2+ and target tyrosine. The model is consistent with an in‐line nucleophilic attack of the deprotonated side‐chain hydroxyl group onto the α‐phosphorus of the nucleotide to accomplish AMP transfer. Furthermore, a general, sequence‐independent mechanism of target positioning through antiparallel β‐strand interactions between enzyme and target is suggested. 相似文献
2.
过去的研究发现了一个具有相同保守结构域的Fic(filamentation induced by cAMP)蛋白家族。虽然在原核生物中发现超过3 000种以上含有Fic结构域的不同蛋白质,但到目前为止,在包括人在内的真核生物中仅发现一种Fic蛋白。Fic结构域主要通过含有磷酸基团的化合物在转录中起作用,目前被人类关注的功能是单酰磷酸化(AMPylation, AMP化),一种类似磷酸化的蛋白质调控机制,是以ATP为底物将一磷酸腺苷(AMP)特异地转移至靶标氨基酸残基。该机制主要在细菌侵袭宿主、侵袭后增殖、致病的过程中发挥作用。真核细胞中的单磷酸腺苷化可以调控内质网的稳定性。本文主要对Fic蛋白的特征性结构和作用以及几种主要的Fic蛋白的结构、作用和研究方法进行综述。 相似文献
3.
A conserved amino acid sequence motif was identified in four distinct groups of enzymes that catalyze the hydrolysis of the α–β phosphate bond of ATP, namely GMP synthetases, argininosuccinate synthetases, asparagine synthetases, and ATP sulfurylases. The motif is also present in Rhodobacter capsulata AdgA, Escherichia coli NtrL, and Bacillus subtilis OutB, for which no enzymatic activities are currently known. The observed pattern of amino acid residue conservation and predicted secondary structures suggest that this motif may be a modified version of the P-loop of nucleotide binding domains, and that it is likely to be involved in phosphate binding. We call it PP-motif, since it appears to be a part of a previously uncharacterized ATP pyrophophatase domain. ATP sulfurylases, NtrL, and OutB consist of this domain alone. In other proteins, the pyrophosphatase domain is associated with amidotransferase domains (type I or type II), a putative citrulline-aspartate ligase domain or a nitrilase/amidase domain. Unexpectedly, statistically significant overall sequence similarity was found between ATP sulfurylase and 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductase, another protein of the sulfate activation pathway. The PP-motif is strongly modified in PAPS reductases, but they share with ATP sulfurylases another conserved motif which might be involved in sulfate binding. We propose that PAPS reductases may have evolved from ATP sulfurylases; the evolution of the new enzymatic function appears to be accompanied by a switch of the strongest functional constraint from the PP-motif to the putative sulfate-binding motif. © 1994 Wiley-Liss, Inc. 相似文献
4.
Investigating the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase using lanthanide luminescence spectroscopy.
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L. P. Reynaldo J. J. Villafranca W. D. Horrocks Jr 《Protein science : a publication of the Protein Society》1996,5(12):2532-2544
Lanthanide luminescence was used to examine the effects of posttranslational adenylylation on the metal binding sites of Escherichia coli glutamine synthetase (GS). These studies revealed the presence of two lanthanide ion binding sites of GS of either adenylylation extrema. Individual emission decay lifetimes were obtained in both H2O and D2O solvent systems, allowing for the determination of the number of water molecules coordinated to each bound Eu3+. The results indicate that there are 4.3 +/- 0.5 and 4.6 +/- 0.5 water molecules coordinated to Eu3+ bound to the n1 site of unadenylylated enzyme, GS0, and fully adenylylated enzyme, GS12, respectively, and that there are 2.6 +/- 0.5 water molecules coordinated to Eu3+ at site n2 for both GS0 and GS12. Energy transfer measurements between the lanthanide donor-acceptor pair Eu3+ and Nd3+, obtained an intermetal distance measurement of 12.1 +/- 1.5 A. Distances between a Tb3+ ion at site n2 and tryptophan residues were also performed with the use of single-tryptophan mutant forms of E. coli GS. The dissociation constant for lanthanide ion binding to site n1 was observed to decrease from Kd = 0.35 +/- 0.09 microM for GS0 to Kd = 0.06 +/- 0.02 microM for GS12. The dissociation constant for lanthanide ion binding to site n2 remained unchanged as a function of adenylylation state; Kd = 3.8 +/- 0.9 microM and Kd = 2.6 +/- 0.7 microM for GS0 and GS12, respectively. Competition experiments indicate that Mn2+ affinity at site n1 decreases as a function of increasing adenylylation state, from Kd = 0.05 +/- 0.02 microM for GS0 to Kd = 0.35 +/- 0.09 microM for GS12. Mn2+ affinity at site n2 remains unchanged (Kd = 5.3 +/- 1.3 microM for GS0 and Kd = 4.0 +/- 1.0 microM for GS12). The observed divalent metal ion affinities, which are affected by the adenylylation state, agrees with other steady-state substrate experiments (Abell LM, Villafranca JJ, 1991, Biochemistry 30:1413-1418), supporting the hypothesis that adenylylation regulates GS by altering substrate and metal ion affinities. 相似文献
5.
The anaerobic acetogenic bacterium Acetobacterium woodii employs a novel type of Na(+)-motive anaerobic respiration, caffeate respiration. However, this respiration is at the thermodynamic limit of energy conservation, and even worse, in the first step, caffeate is activated by caffeyl-CoA synthetase, which hydrolyzes ATP to AMP and pyrophosphate. Here, we have addressed whether or not the energy stored in the anhydride bond of pyrophosphate is conserved by A. woodii. Inverted membrane vesicles of A. woodii have a membrane-bound pyrophosphatase that catalyzes pyrophosphate hydrolysis at a rate of 70-120 milliunits/mg of protein. Pyrophosphatase activity was dependent on the divalent cation Mg(2+). In addition, activity was strictly dependent on Na(+) with a K(m) of 1.1 mM. Hydrolysis of pyrophosphate was accompanied by (22)Na(+) transport into the lumen of the inverted membrane vesicles. Inhibitor studies revealed that (22)Na(+) transport was primary and electrogenic. Next to the Na(+)-motive ferredoxin:NAD(+) oxidoreductase (Fno or Rnf), the Na(+)-pyrophosphatase is the second primary Na(+)-translocating enzyme in A. woodii. 相似文献
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7.
Viruses are obligatory parasites that can replicate only inside host cells. Therefore, the evolutionary drive to enter cells is immense, leading to diversification in the cell-entry strategies of viruses. One of the most critical steps for cell entry is the recognition of the target cell, a process driven by the formation of viral/host macromolecular complexes. The accumulation of recent structural data for viruses within the arenaviridae family allows us to examine how different viral species from the same viral family utilize evolutionarily-related viral glycoproteins to engage with a variety of different cellular receptors. These structural data, compared to other viruses from the coronaviridae family, hint about possible routes that such viruses use for evolving new receptor-binding capabilities, allowing them to switch from one receptor to another. 相似文献
8.
Candy A. Heyen 《Biochemical and biophysical research communications》2009,390(4):1414-787
Recombinant mouse UDP-glucose pyrophosphatase (UGPPase), encoded by the Nudt14 gene, was produced in Escherichia coli and purified close to homogeneity. The enzyme catalyzed the conversion of [β-32P]UDP-glucose to [32P]glucose-1-P and UMP, confirming that it hydrolyzed the pyrophosphate of the nucleoside diphosphate sugar to generate glucose-1-P and UMP. The enzyme was also active toward ADP-ribose. Activity is dependent on the presence of Mg2+ and was greatest at alkaline pH above 8. Kinetic analysis indicated a Km of ∼4 mM for UDP-glucose and ∼0.3 mM for ADP-ribose. Based on Vmax/Km values, the enzyme was ∼20-fold more active toward ADP-ribose. UGPPase behaves as a dimer in solution and can be cross-linked to generate a species of Mr 54,000 from a monomer of 30,000 as judged by SDS-PAGE. The dimerization was not affected by the presence of glucose-1-P or UDP-glucose. Using antibodies raised against the recombinant protein, Western analysis indicated that UGPPase was widely expressed in mouse tissues, including skeletal muscle, liver, kidney, heart, lung, fat, heart and pancreas with a lower level in brain. It was generally present as a doublet when analyzed by SDS-PAGE, suggesting the occurrence of some form of post-translational modification. Efforts to interconvert the species by adding or inhibiting phosphatase activity were unsuccessful, leaving the nature of the modification unknown. Sequence alignments and database searches revealed related proteins in species as distant as Drosophila melanogaster and Caenorhabditis elegans. 相似文献
9.
Grycova L Lansky Z Friedlova E Vlachova V Kubala M Obsilova V Obsil T Teisinger J 《Archives of biochemistry and biophysics》2007,465(2):389-398
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labelling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations. 相似文献
10.
Despite significant fluctuation under thermal noise, biological machines in cells perform their tasks with exquisite precision. Using molecular simulation of a coarse-grained model and theoretical arguments, we envisaged how kinesin, a prototype of biological machines, generates force and regulates its dynamics to sustain persistent motor action. A structure-based model, which can be versatile in adapting its structure to external stresses while maintaining its native fold, was employed to account for several features of kinesin dynamics along the biochemical cycle. This analysis complements our current understandings of kinesin dynamics and connections to experiments. We propose a thermodynamic cycle for kinesin that emphasizes the mechanical and regulatory role of the neck linker and clarify issues related to the motor directionality, and the difference between the external stalling force and the internal tension responsible for the head-head coordination. The comparison between the thermodynamic cycle of kinesin and macroscopic heat engines highlights the importance of structural change as the source of work production in biomolecular machines. 相似文献
11.
Pedone E D'Ambrosio K De Simone G Rossi M Pedone C Bartolucci S 《Journal of molecular biology》2006,356(1):155-164
A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (PDI)-like proteins. Members of this family are characterized by a molecular mass of about 26kDa and by two Trx folds, each comprising a CXXC active site motif. We report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO). Functional studies have revealed the high catalytic efficiency of this enzyme in reducing, oxidizing and isomerizing disulfide bridges. Site-directed mutagenesis experiments have suggested that its two active sites have similar functional properties, i.e. that each of them imparts partial activity to the enzyme. This similarity was confirmed by the analysis of the enzyme crystal structure, which points to similar geometrical parameters and solvent accessibilities for the two active sites. The results demonstrated that AaPDO is the most PDI-like of all prokaryotic proteins so far known. Thus, further experimental studies on this enzyme are likely to provide important information on the eukaryotic homologue. 相似文献
12.
13.
In the era of structural genomics, it is necessary to generate accurate structural alignments in order to build good templates for homology modeling. Although a great number of structural alignment algorithms have been developed, most of them ignore intermolecular interactions during the alignment procedure. Therefore, structures in different oligomeric states are barely distinguishable, and it is very challenging to find correct alignment in coil regions. Here we present a novel approach to structural alignment using a clique finding algorithm and environmental information (SAUCE). In this approach, we build the alignment based on not only structural coordinate information but also realistic environmental information extracted from biological unit files provided by the Protein Data Bank (PDB). At first, we eliminate all environmentally unfavorable pairings of residues. Then we identify alignments in core regions via a maximal clique finding algorithm. Two extreme value distribution (EVD) form statistics have been developed to evaluate core region alignments. With an optional extension step, global alignment can be derived based on environment-based dynamic programming linking. We show that our method is able to differentiate three-dimensional structures in different oligomeric states, and is able to find flexible alignments between multidomain structures without predetermined hinge regions. The overall performance is also evaluated on a large scale by comparisons to current structural classification databases as well as to other alignment methods. 相似文献
14.
Lahmy S Barnèche F Derancourt J Filipowicz W Delseny M Echeverria M 《FEBS letters》2000,480(2-3):255-260
P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing. 相似文献
15.
A structural perspective on ClC channel and transporter function 总被引:1,自引:0,他引:1
Dutzler R 《FEBS letters》2007,581(15):2839-2844
The ClC chloride channels and transporters constitute a large family of membrane proteins that is involved in a variety of physiological processes. All members share a conserved molecular architecture that consists of a complex transmembrane transport domain followed by a cytoplasmic domain. Despite the strong conservation, the family shows an unusually broad variety of functional behaviors as some members work as gated chloride channels and others as secondary active chloride transporters. The conservation in the structure and the functional resemblance of gating and coupled transport suggests a strong mechanistic relationship between these seemingly contradictory transport modes. The cytoplasmic domains constitute putative regulatory components that are ubiquitous in eukaryotic ClC family members and that in certain cases interact with nucleotides thus linking ion transport to nucleotide sensing by yet unknown mechanisms. 相似文献
16.
17.
Caputi L Malnoy M Goremykin V Nikiforova S Martens S 《The Plant journal : for cell and molecular biology》2012,69(6):1030-1042
For almost a decade, our knowledge on the organisation of the family 1 UDP‐glycosyltransferases (UGTs) has been limited to the model plant A. thaliana. The availability of other plant genomes represents an opportunity to obtain a broader view of the family in terms of evolution and organisation. Family 1 UGTs are known to glycosylate several classes of plant secondary metabolites. A phylogeny reconstruction study was performed to get an insight into the evolution of this multigene family during the adaptation of plants to life on land. The organisation of the UGTs in the different organisms was also investigated. More than 1500 putative UGTs were identified in 12 fully sequenced and assembled plant genomes based on the highly conserved PSPG motif. Analyses by maximum likelihood (ML) method were performed to reconstruct the phylogenetic relationships existing between the sequences. The results of this study clearly show that the UGT family expanded during the transition from algae to vascular plants and that in higher plants the clustering of UGTs into phylogenetic groups appears to be conserved, although gene loss and gene gain events seem to have occurred in certain lineages. Interestingly, two new phylogenetic groups, named O and P, that are not present in A. thaliana were discovered. 相似文献
18.
Rich PR 《Journal of bioenergetics and biomembranes》2008,40(5):407-410
In 1991 Peter Mitchell wrote a last article that summarised his views on the origin, development and current status of his chemiosmotic ideas. I here review some of his views of that time on structures and mechanisms of several key bioenergetic components in relation to the subsequent advances that have been made. 相似文献
19.
M. Joanne Lemieux 《生物化学与生物物理学报:生物膜》2008,1778(9):1805-1813
Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists. 相似文献
20.
Lemieux MJ 《Biochimica et biophysica acta》2008,1778(9):1805-1813
Electrochemical potential-driven transporters represent a vast array of proteins with varied substrate specificities. While diverse in size and substrate specificity, they are all driven by electrochemical potentials. Over the past five years there have been increasing numbers of X-ray structures reported for this family of transporters. Structural information is available for five subfamilies of electrochemical potential-driven transporters. No structural information exists for the remaining 91 subfamilies. In this review, the various subfamilies of electrochemical potential-driven transporters are discussed. The seven reported structures for the electrochemical potential-driven transporters and the methods for their crystallization are also presented. With a few exceptions, overall crystallization trends have been very similar for the transporters despite their differences in substrate specificity and topology. Also discussed is why the structural studies on these transporters were successful while others are not as fruitful. With the plethora of transporters with unknown structures, this review provides incentive for crystallization of transporters in the remaining subfamilies for which no structural information exists. 相似文献