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1.  CRISPR RNA binding and DNA target recognition by purified Cascade complexes from Escherichia coli 
Nucleic Acids Research  2014;43(1):530-543.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated Cas proteins comprise a prokaryotic RNA-guided adaptive immune system that interferes with mobile genetic elements, such as plasmids and phages. The type I-E CRISPR interference complex Cascade from Escherichia coli is composed of five different Cas proteins and a 61-nt-long guide RNA (crRNA). crRNAs contain a unique 32-nt spacer flanked by a repeat-derived 5′ handle (8 nt) and a 3′ handle (21 nt). The spacer part of crRNA directs Cascade to DNA targets. Here, we show that the E. coli Cascade can be expressed and purified from cells lacking crRNAs and loaded in vitro with synthetic crRNAs, which direct it to targets complementary to crRNA spacer. The deletion of even one nucleotide from the crRNA 5′ handle disrupted its binding to Cascade and target DNA recognition. In contrast, crRNA variants with just a single nucleotide downstream of the spacer part bound Cascade and the resulting ribonucleotide complex containing a 41-nt-long crRNA specifically recognized DNA targets. Thus, the E. coli Cascade-crRNA system exhibits significant flexibility suggesting that this complex can be engineered for applications in genome editing and opening the way for incorporation of site-specific labels in crRNA.
doi:10.1093/nar/gku1285
PMCID: PMC4288178  PMID: 25488810
2.  Toroidal structure and DNA cleavage by the CRISPR-associated [4Fe-4S]-cluster containing Cas4 nuclease SSO0001 from Sulfolobus solfataricus 
Journal of the American Chemical Society  2013;135(46):17476-17487.
Cas4 proteins, a core protein family associated with the microbial system of adaptive immunity CRISPR, are predicted to function in the adaptation step of the CRISPR mechanism. Here we show that the Cas4 protein SSO0001 from the archaeon Sulfolobus solfataricus has metal-dependent endonuclease and 5' to 3' exonuclease activities against single-stranded DNA, as well as ATP-independent DNA unwinding activity toward double-stranded DNA. The crystal structure of SSO0001 revealed a decameric toroid formed by five dimers with each protomer containing one [4Fe-4S] cluster and one Mn2+ ion bound in the active site located inside the internal tunnel. The conserved RecB motif and four Cys residues are important for DNA binding and cleavage activities, whereas DNA unwinding depends on several residues located near the [4Fe-4S]-cluster. Our results suggest that Cas4 proteins might contribute to the addition of novel CRISPR spacers through the formation of 3'-DNA overhangs and to the degradation of foreign DNA.
doi:10.1021/ja408729b
PMCID: PMC3889865  PMID: 24171432
CRISPR interference; Cas4; exonuclease; RecB motif; [4Fe-4S] cluster
3.  The CRISPR-associated Cas4 protein Pcal_0546 from Pyrobaculum calidifontis contains a [2Fe-2S] cluster: crystal structure and nuclease activity 
Nucleic Acids Research  2014;42(17):11144-11155.
Cas4 nucleases constitute a core family of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated proteins, but little is known about their structure and activity. Here we report the crystal structure of the Cas4 protein Pcal_0546 from Pyrobaculum calidifontis, which revealed a monomeric protein with a RecB-like fold and one [2Fe-2S] cluster coordinated by four conserved Cys residues. Pcal_0546 exhibits metal-dependent 5′ to 3′ exonuclease activity against ssDNA substrates, whereas the Cas4 protein SSO1391 from Sulfolobus solfataricus can cleave ssDNA in both the 5′ to 3′ and 3′ to 5′ directions. The active site of Pcal_0546 contains a bound metal ion coordinated by the side chains of Asp123, Glu136, His146, and the main chain carbonyl of Ile137. Site-directed mutagenesis of Pcal_0546 and SSO1391 revealed that the residues of RecB motifs II, III and QhXXY are critical for nuclease activity, whereas mutations of the conserved Cys residues resulted in a loss of the iron-sulfur cluster, but had no effect on DNA cleavage. Our results revealed the biochemical diversity of Cas4 nucleases, which can have different oligomeric states, contain [4Fe-4S] or [2Fe-2S] clusters, and cleave single stranded DNA in different directions producing single-stranded DNA overhangs, which are potential intermediates for the synthesis of new CRISPR spacers.
doi:10.1093/nar/gku797
PMCID: PMC4176176  PMID: 25200083
4.  Structure and activity of the cold-active and anion-activated carboxyl esterase OLEI01171 from the oil-degrading marine bacterium Oleispira antarctica 
The Biochemical journal  2012;445(2):193-203.
The uncharacterized α/β-hydrolase protein OLEI01171 from the psychrophilic marine bacterium Oleispira antarctica belongs to the PF00756 family of putative esterases, which also includes human esterase D. In the present paper we show that purified recombinant OLEI01171 exhibits high esterase activity against the model esterase substrate α-naphthyl acetate at 5 – 30°C with maximal activity at 15–20°C. The esterase activity of OLEI01171 was stimulated 3–8-fold by the addition of chloride or several other anions (0.1–1.0 M). Compared with mesophilic PF00756 esterases, OLEI01171 exhibited a lower overall protein thermostability. Two crystal structures ofOLEI01171 were solved at 1.75 and 2.1 Å resolution and revealed a classical serine hydrolase catalytic triad and the presence of a chloride or bromide ion bound in the active site close to the catalytic Ser148.Both anions were found to co-ordinate a potential catalytic water molecule located in the vicinity of the catalytic triad His257. The results of the present study suggest that the bound anion perhaps contributes to the polarization of the catalytic water molecule and increases the rate of the hydrolysis of an acyl-enzyme intermediate. Alanine replacement mutagenesis of OLEI01171 identified ten amino acid residues important for esterase activity. The replacement of Asn225 by lysine had no significant effect on the activity or thermostability of OLEI01171, but resulted in a detectable increase of activity at 35–45°C. The present study has provided insight into the molecular mechanisms of activity of a cold-active and anion-activated carboxyl esterase.
doi:10.1042/BJ20112113
PMCID: PMC4127636  PMID: 22519667
anion activation; carboxyl esterase; cold-active enzyme; crystal structure; Oleispira antarctica; protein thermostability
5.  Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801 
The Biochemical journal  2012;444(3):10.1042/BJ20112032.
The hotdog fold is one of the basic protein folds widely present in bacteria, archaea, and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signaling, and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (1.70 and 1.75 Å) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long alpha helix (Asp57 in PA5202 and Glu35 in PA2801). Alanine replacement mutagenesis of PA5202 identified four residues (Asn42, Arg43, Asp57, and Thr76), which are critical for activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis suggesting a functional link between the PA5202 activity and pyocyanin production. Thus, the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.
doi:10.1042/BJ20112032
PMCID: PMC3836677  PMID: 22439787
hotdog fold; thioesterase; crystal structure; pyocyanin; Pseudomonas aeruginosa
6.  Biochemical and Structural Studies of Conserved Maf Proteins Revealed Nucleotide Pyrophosphatases with a Preference for Modified Nucleotides 
Chemistry & Biology  2013;20(11):1386-1398.
Summary
Maf (for multicopy associated filamentation) proteins represent a large family of conserved proteins implicated in cell division arrest but whose biochemical activity remains unknown. Here, we show that the prokaryotic and eukaryotic Maf proteins exhibit nucleotide pyrophosphatase activity against 5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP, which represent the most abundant modified bases in all organisms, as well as against canonical nucleotides dTTP, UTP, and CTP. Overexpression of the Maf protein YhdE in E. coli cells increased intracellular levels of dTMP and UMP, confirming that dTTP and UTP are the in vivo substrates of this protein. Crystal structures and site-directed mutagenesis of Maf proteins revealed the determinants of their activity and substrate specificity. Thus, pyrophosphatase activity of Maf proteins toward canonical and modified nucleotides might provide the molecular mechanism for a dual role of these proteins in cell division arrest and house cleaning.
Graphical Abstract
Highlights
•Maf proteins represent a family of nucleoside triphosphate pyrophosphatases•Maf proteins hydrolyze the canonical nucleotides dTTP, UTP, and CTP•Maf proteins are also active against m5UTP, m5CTP, pseudo-UTP, and m7GTP•Maf structures reveal the molecular mechanisms of their substrate selectivity
Tchigvintsev et al. show that Maf proteins are a family of nucleotide pyrophosphatases active against both canonical and modified nucleotides. This suggests that Mafs might have a dual role in cell division and in the prevention of the incorporation of modified nucleotides into cellular nucleic acids.
doi:10.1016/j.chembiol.2013.09.011
PMCID: PMC3899018  PMID: 24210219
7.  Biochemical studies of the multicopper oxidase (small laccase) from Streptomyces coelicolor using bioactive phytochemicals and site-directed mutagenesis 
Microbial Biotechnology  2013;6(5):588-597.
Summary
Multicopper oxidases can act on a broad spectrum of phenolic and non-phenolic compounds. These enzymes include laccases, which are widely distributed in plants and fungi, and were more recently identified in bacteria. Here, we present the results of biochemical and mutational studies of small laccase (SLAC), a multicopper oxidase from Streptomyces coelicolor (SCO6712). In addition to typical laccase substrates, SLAC was tested using phenolic compounds that exhibit antioxidant activity. SLAC showed oxidase activity against 12 of 23 substrates tested, including caffeic acid, ferulic acid, resveratrol, quercetin, morin, kaempferol and myricetin. The kinetic parameters of SLAC were determined for 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid), 2,6-dimethoxyphenol, quercetin, morin and myricetin, and maximum reaction rates were observed with myricetin, where kcat and Km values at 60°C were 8.1 (± 0.8) s−1 and 0.9 (± 0.3) mM respectively. SLAC had a broad pH optimum for activity (between pH 4 and 8) and temperature optimum at 60–70°C. It demonstrated remarkable thermostability with a half-life of over 10 h at 80°C and over 7 h at 90°C. Site-directed mutagenesis revealed 17 amino acid residues important for SLAC activity including the 10 His residues involved in copper coordination. Most notably, the Y229A and Y230A mutant proteins showed over 10-fold increase in activity compared with the wild-type SLAC, which was correlated to higher copper incorporation, while kinetic analyses with S929A predicts localization of this residue near the meta-position of aromatic substrates.
Funding Information Funding for this research was provided by the Government of Ontario for the project ‘FFABnet: Functionalized Fibre and Biochemicals’ (ORF-RE-05-005), and the Natural Sciences and Engineering Research Council of Canada.
doi:10.1111/1751-7915.12068
PMCID: PMC3918160  PMID: 23815400
8.  Discovery and validation of gene classifiers for endocrine-disrupting chemicals in zebrafish (danio rerio) 
BMC Genomics  2012;13:358.
Background
Development and application of transcriptomics-based gene classifiers for ecotoxicological applications lag far behind those of biomedical sciences. Many such classifiers discovered thus far lack vigorous statistical and experimental validations. A combination of genetic algorithm/support vector machines and genetic algorithm/K nearest neighbors was used in this study to search for classifiers of endocrine-disrupting chemicals (EDCs) in zebrafish. Searches were conducted on both tissue-specific and tissue-combined datasets, either across the entire transcriptome or within individual transcription factor (TF) networks previously linked to EDC effects. Candidate classifiers were evaluated by gene set enrichment analysis (GSEA) on both the original training data and a dedicated validation dataset.
Results
Multi-tissue dataset yielded no classifiers. Among the 19 chemical-tissue conditions evaluated, the transcriptome-wide searches yielded classifiers for six of them, each having approximately 20 to 30 gene features unique to a condition. Searches within individual TF networks produced classifiers for 15 chemical-tissue conditions, each containing 100 or fewer top-ranked gene features pooled from those of multiple TF networks and also unique to each condition. For the training dataset, 10 out of 11 classifiers successfully identified the gene expression profiles (GEPs) of their targeted chemical-tissue conditions by GSEA. For the validation dataset, classifiers for prochloraz-ovary and flutamide-ovary also correctly identified the GEPs of corresponding conditions while no classifier could predict the GEP from prochloraz-brain.
Conclusions
The discrepancies in the performance of these classifiers were attributed in part to varying data complexity among the conditions, as measured to some degree by Fisher’s discriminant ratio statistic. This variation in data complexity could likely be compensated by adjusting sample size for individual chemical-tissue conditions, thus suggesting a need for a preliminary survey of transcriptomic responses before launching a full scale classifier discovery effort. Classifier discovery based on individual TF networks could yield more mechanistically-oriented biomarkers. GSEA proved to be a flexible and effective tool for application of gene classifiers but a similar and more refined algorithm, connectivity mapping, should also be explored. The distribution characteristics of classifiers across tissues, chemicals, and TF networks suggested a differential biological impact among the EDCs on zebrafish transcriptome involving some basic cellular functions.
doi:10.1186/1471-2164-13-358
PMCID: PMC3469349  PMID: 22849515
Gene classifiers; Endocrine-disrupting chemicals; Transcriptomics; Mechanism of action; Zebrafish
9.  A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair 
Molecular microbiology  2010;79(2):484-502.
Summary
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks, and 5′-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.
doi:10.1111/j.1365-2958.2010.07465.x
PMCID: PMC3071548  PMID: 21219465
Cas1; CRISPR; DNA recombination; DNA repair; nuclease; YgbT
10.  Structural insight into the mechanism of cyclic di-GMP hydrolysis by EAL domain phosphodiesterases 
Journal of molecular biology  2010;402(3):524-538.
Cyclic diguanylate (c-di-GMP) is a ubiquitous second messenger regulating diverse cellular functions including motility, biofilm formation, cell cycle progression and virulence in bacteria. In the cell, degradation of c-di-GMP is catalyzed by highly specific EAL domain phosphodiesterases whose catalytic mechanism is still unclear. Here, we purified 13 EAL domain proteins from various organisms and demonstrated that their catalytic activity is associated with the presence of 10 conserved EAL domain residues. The crystal structure of the TDB1265 EAL domain was determined in a free state (1.8 Å) and in complex with c-di-GMP (2.35 Å) and unveiled the role of the conserved residues in substrate binding and catalysis. The structure revealed the presence of two metal ions directly coordinated by six conserved residues, two oxygens of the c-di-GMP phosphate, and potential catalytic water molecule. Our results support a two-metal-ion catalytic mechanism of c-di-GMP hydrolysis by EAL domain phosphodiesterases.
doi:10.1016/j.jmb.2010.07.050
PMCID: PMC2945410  PMID: 20691189
EAL domain; cyclic di-GMP; phosphodiesterase; X-ray crystallography; Thiobacillus denitrificans
11.  Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica 
Nature Communications  2013;4:2156.
Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis—the paradigm of mesophilic hydrocarbonoclastic bacteria—O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
Oleispira antarctica is an oil-degrading bacterium found in the cold and deep sea. Here Kube et al. report the genome sequence of O. antarctica and provide a comprehensive functional genetic and protein structural analysis, revealing insights into how this organism has adapted to its cold environment.
doi:10.1038/ncomms3156
PMCID: PMC3759055  PMID: 23877221

Results 1-11 (11)