Fusion of a functional glutaredoxin to the radical-generating subunit of ribonucleotide reductase

AbstractClass I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyse reduction of the four ribonucleotides to their corresponding deoxyribonucleotides.Facklamia ignavaNrdB is an unprecedented fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-cone. Grx, which in general is encoded elsewhere in the genome than is the RNR operon, is a known physiological reductant of RNRs. Here we show that the fused Grx domain functions as an efficient reductant of theF. ignavaclass I RNR via the common dithiol mechanism and interestingly also via a monothiol mechanism, although less efficiently. A Grx that utilizes either or of these two reaction mechanisms has to our knowledge not been observed with a native substrate before. The ATP-cone, which is commonly found as an N-terminal domain of the catalytic subunit of RNRs, is an allosteric on/off switch that promotes dNDP reduction in presence of ATP and inhibits the enzyme activity in presence of dATP. Here we show that dATP bound to the ATP-cone ofF. ignavaNrdB promotes formation of tetramers that are unable to form enzymatically competent complexes withF. ignavaNrdA. The ATP-cone binds two molecules of dATP, but only one molecule of the activating nucleotide ATP.F. ignavaNrdB contains the recently identified radical factor Mn2III/IV. We show that NrdA from the firmicuteF. ignavacan form a catalytically competent RNR with the Mn2III/IV-containing NrdB from the flavobacteriumLeeuwenhoekiella blandensis.

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A glutaredoxin domain fused to the radical-generating subunit of ribonucleotide reductase (RNR) functions as an efficient RNR reductant

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.004991 ◽

2018 ◽

Vol 293 (41) ◽

pp. 15889-15900 ◽

Cited By ~ 7

Author(s):

Inna Rozman Grinberg ◽

Daniel Lundin ◽

Margareta Sahlin ◽

Mikael Crona ◽

Gustav Berggren ◽

...

Keyword(s):

Fusion Protein ◽

Ribonucleotide Reductase ◽

Reaction Mechanisms ◽

Binding Domain ◽

Catalytic Subunit ◽

Class I ◽

Atp Binding ◽

Terminal Domain ◽

The Common

Class I ribonucleotide reductase (RNR) consists of a catalytic subunit (NrdA) and a radical-generating subunit (NrdB) that together catalyze reduction of ribonucleotides to their corresponding deoxyribonucleotides. NrdB from the firmicute Facklamia ignava is a unique fusion protein with N-terminal add-ons of a glutaredoxin (Grx) domain followed by an ATP-binding domain, the ATP cone. Grx, usually encoded separately from the RNR operon, is a known RNR reductant. We show that the fused Grx domain functions as an efficient reductant of the F. ignava class I RNR via the common dithiol mechanism and, interestingly, also via a monothiol mechanism, although less efficiently. To our knowledge, a Grx that uses both of these two reaction mechanisms has not previously been observed with a native substrate. The ATP cone is in most RNRs an N-terminal domain of the catalytic subunit. It is an allosteric on/off switch promoting ribonucleotide reduction in the presence of ATP and inhibiting RNR activity in the presence of dATP. We found that dATP bound to the ATP cone of F. ignava NrdB promotes formation of tetramers that cannot form active complexes with NrdA. The ATP cone bound two dATP molecules but only one ATP molecule. F. ignava NrdB contains the recently identified radical-generating cofactor MnIII/MnIV. We show that NrdA from F. ignava can form a catalytically competent RNR with the MnIII/MnIV-containing NrdB from the flavobacterium Leeuwenhoekiella blandensis. In conclusion, F. ignava NrdB is fused with a Grx functioning as an RNR reductant and an ATP cone serving as an on/off switch.

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Interactions between the Escherichia coli cAMP receptor protein and the C-terminal domain of the α subunit of RNA polymerase at Class I promoters

Biochemical Journal ◽

10.1042/bj3370415 ◽

1999 ◽

Vol 337 (3) ◽

pp. 415-423 ◽

Cited By ~ 8

Author(s):

Emma C. LAW ◽

Nigel J. SAVERY ◽

Stephen J. W. BUSBY

Keyword(s):

Escherichia Coli ◽

Rna Polymerase ◽

Transcription Initiation ◽

Class I ◽

Receptor Protein ◽

Camp Receptor Protein ◽

Α Subunit ◽

Terminal Domain ◽

Up Elements ◽

Camp Receptor

The Escherichia coli cAMP receptor protein (CRP) is a factor that activates transcription at over 100 target promoters. At Class I CRP-dependent promoters, CRP binds immediately upstream of RNA polymerase and activates transcription by making direct contacts with the C-terminal domain of the RNA polymerase α subunit (αCTD). Since αCTD is also known to interact with DNA sequence elements (known as UP elements), we have constructed a series of semi-synthetic Class I CRP-dependent promoters, carrying both a consensus DNA-binding site for CRP and a UP element at different positions. We previously showed that, at these promoters, the CRP–αCTD interaction and the CRP–UP element interaction contribute independently and additively to transcription initiation. In this study, we show that the two halves of the UP element can function independently, and that, in the presence of the UP element, the best location for the DNA site for CRP is position -69.5. This suggests that, at Class I CRP-dependent promoters where the DNA site for CRP is located at position -61.5, the two αCTDs of RNA polymerase are not optimally positioned. Two experiments to test this hypothesis are presented.

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Anti-Cdc25 antibodies inhibit guanyl nucleotide-dependent adenylyl cyclase of Saccharomyces cerevisiae and cross-react with a 150-kilodalton mammalian protein

Molecular and Cellular Biology ◽

10.1128/mcb.12.6.2653-2661.1992 ◽

1992 ◽

Vol 12 (6) ◽

pp. 2653-2661

Author(s):

E Gross ◽

I Marbach ◽

D Engelberg ◽

M Segal ◽

G Simchen ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Fusion Protein ◽

Adenylyl Cyclase ◽

Gene Product ◽

Cyclase Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Terminal Domain ◽

Yeast Homolog ◽

Cdc25 Protein ◽

Beta Galactosidase

The CDC25 gene product of the yeast Saccharomyces cerevisiae has been shown to be a positive regulator of the Ras protein. The high degree of homology between yeast RAS and the mammalian proto-oncogene ras suggests a possible resemblance between the mammalian regulator of Ras and the regulator of the yeast Ras (Cdc25). On the basis of this assumption, we have raised antibodies against the conserved C-terminal domain of the Cdc25 protein in order to identify its mammalian homologs. Anti-Cdc25 antibodies raised against a beta-galactosidase-Cdc25 fusion protein were purified by immunoaffinity chromatography and were shown by immunoblotting to specifically recognize the Cdc25 portion of the antigen and a truncated Cdc25 protein, also expressed in bacteria. These antibodies were shown both by immunoblotting and by immunoprecipitation to recognize the CDC25 gene product in wild-type strains and in strains overexpressing Cdc25. The anti-Cdc25 antibodies potently inhibited the guanyl nucleotide-dependent and, approximately 3-fold less potently, the Mn(2+)-dependent adenylyl cyclase activity in S. cerevisiae. The anti-Cdc25 antibodies do not inhibit cyclase activity in a strain harboring RAS2Val-19 and lacking the CDC25 gene product. These results support the view that Cdc25, Ras2, and Cdc35/Cyr1 proteins are associated in a complex. Using these antibodies, we were able to define the conditions to completely solubilize the Cdc25 protein. The results suggest that the Cdc25 protein is tightly associated with the membrane but is not an intrinsic membrane protein, since only EDTA at pH 12 can solubilize the protein. The anti-Cdc25 antibodies strongly cross-reacted with the C-terminal domain of the Cdc25 yeast homolog, Sdc25. Most interestingly, these antibodies also cross-reacted with mammalian proteins of approximately 150 kDa from various tissues of several species of animals. These interactions were specifically blocked by the beta-galactosidase-Cdc25 fusion protein.

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The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽

10.1242/dev.126.13.2841 ◽

1999 ◽

Vol 126 (13) ◽

pp. 2841-2853 ◽

Cited By ~ 1

Author(s):

G. Deshpande ◽

G. Calhoun ◽

P.D. Schedl

Keyword(s):

Fusion Protein ◽

Rna Binding ◽

Chimeric Protein ◽

Dominant Negative ◽

Terminal Domain ◽

Binding Domains ◽

Transcriptional Regulatory ◽

Dominant Negative Activity ◽

Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

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Crystal structure of the N-terminal domain of VqsR fromPseudomonas aeruginosaat 2.1 Å resolution

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x17009025 ◽

2017 ◽

Vol 73 (7) ◽

pp. 431-436

Author(s):

Qing He ◽

Kang Wang ◽

Tiantian Su ◽

Feng Wang ◽

Lichuan Gu ◽

...

Keyword(s):

Crystal Structure ◽

Sequence Similarity ◽

Transcriptional Regulator ◽

Binding Domain ◽

Structural Comparison ◽

Homoserine Lactones ◽

Terminal Domain ◽

C Terminus ◽

Common Motif ◽

The Common

VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las,rhlandpqs) by directly downregulating the expression ofqscRinPseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix–turn–helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1–193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α–β–α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.

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Identification of Rab6 as an N-ethylmaleimide-sensitive fusion protein-binding protein

Biochemical Journal ◽

10.1042/bj3520165 ◽

2000 ◽

Vol 352 (1) ◽

pp. 165-173 ◽

Cited By ~ 14

Author(s):

Sang Yeul HAN ◽

Dong Yoon PARK ◽

Sang Dai PARK ◽

Seung Hwan HONG

Keyword(s):

Fusion Protein ◽

Atpase Activity ◽

Atp Hydrolysis ◽

Polyclonal Antibodies ◽

Binding Protein ◽

Vesicular Transport ◽

Brain Extract ◽

Terminal Domain ◽

Protein Receptor ◽

And Function

In this study we show the interaction of N-ethylmaleimide-sensitive fusion protein (NSF) with a small GTP-binding protein, Rab6. NSF is an ATPase involved in the vesicular transport within eukaryotic cells. Using the yeast two-hybrid system, we have isolated new NSF-binding proteins from the rat lung cDNA library. One of them was Rab6, which is involved in the vesicular transport within the Golgi and trans-Golgi network as a Ras-like GTPase. We demonstrated that the N-terminal domain of NSF interacted with the C-terminal domain of Rab6, and these proteins were co-immunoprecipitated from the rat brain extract. This interaction was maintained preferentially in the presence of hydrolysable ATP. Recombinant NSF-His6 can also bind to C-terminal Rab6–glutathione S-transferase under the conditions to allow the ATP hydrolysis. Surprisingly, Rab6 stimulates the ATPase activity of NSF by approx. 2-fold as does α-soluble NSF attachment protein receptor. Anti-Rab6 polyclonal antibodies significantly inhibited the Rab6-stimulated ATPase activity of NSF. Furthermore, we found that Rab3 and Rab4 can also associate with NSF and stimulate its ATPase activity. Taken together, we propose a model in which Rab can form an ATP hydrolysis-regulated complex with NSF, and function as a signalling molecule to deliver the signal of vesicle fusion through the interaction with NSF.

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Structural and biochemical characterization of bifunctional XynA

10.1101/2020.10.20.348094 ◽

2020 ◽

Author(s):

Wei Xie ◽

Qi Yu ◽

Yun Liu ◽

Ruoting Cao ◽

Ruiqing Zhang ◽

...

Keyword(s):

Crystal Structure ◽

Enzyme Activity ◽

Catalytic Mechanism ◽

Biochemical Characterization ◽

Cellulase Activity ◽

Cost Savings ◽

Site Directed Mutagenesis ◽

Enzyme Activity Assay ◽

Great Cost ◽

Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

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AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

The Journal of Cell Biology ◽

10.1083/jcb.140.5.1055 ◽

1998 ◽

Vol 140 (5) ◽

pp. 1055-1062 ◽

Cited By ~ 248

Author(s):

Alexandre Benmerah ◽

Christophe Lamaze ◽

Bernadette Bègue ◽

Sandra L. Schmid ◽

Alice Dautry-Varsat ◽

...

Keyword(s):

Plasma Membrane ◽

Fusion Protein ◽

Binding Sites ◽

Fluorescent Protein ◽

Coated Vesicle ◽

Mutant Form ◽

A431 Cells ◽

Coated Pits ◽

Receptor Mediated Endocytosis ◽

Terminal Domain

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

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