scholarly journals The YcnI protein from Bacillus subtilis contains a copper-binding domain

2021 ◽  
Author(s):  
Madhura S Damle ◽  
Stephen C Peters ◽  
Veronika A Szalai ◽  
Oriana S. Fisher
Keyword(s):  
Bacillus Subtilis ◽  
Inductively Coupled Plasma ◽  
Copper Ions ◽  
Bacterial Species ◽  
Copper Homeostasis ◽  
Copper Chaperone ◽  
Copper Binding ◽  
Bioinformatics Analyses ◽  
Paramagnetic Resonance ◽  
X Ray Crystallography

Bacteria require a precise balance of copper ions to ensure that essential cuproproteins are fully metalated while also avoiding copper-induced toxicity. The Gram positive bacterium Bacillus subtilis maintains appropriate copper homeostasis in part through its ycn operon. The ycn operon comprises genes encoding three proteins: the putative copper importer YcnJ, the copper-dependent transcriptional repressor YcnK, and the uncharacterized DUF1775 domain-containing YcnI. DUF1775 domains are found across bacterial phylogeny and bioinformatics analyses indicate that they frequently neighbor domains implicated in copper homeostasis and transport. Here, we investigated whether YcnI can interact with copper and, using electron paramagnetic resonance (EPR) and inductively-coupled plasma-mass spectrometry (ICP-MS) find that it can bind a single Cu(II) ion. We determine the structure of both the apo and copper-bound forms of the protein by X-ray crystallography, uncovering a copper binding site featuring a unique mono-histidine brace ligand set that is highly conserved among DUF1775 domains. These data suggest a possible role for YcnI as a copper chaperone and that DUF1775 domains in other bacterial species may also function in copper homeostasis.

scholarly journals Copper-mediated dimerization of CopZ, a predicted copper chaperone from Bacillus subtilis

2002 ◽  
Vol 368 (3) ◽  
pp. 729-739 ◽  
Author(s):  
Margaret A. KIHLKEN ◽  
Andrew P. LEECH ◽  
Nick E. LE BRUN
Keyword(s):  
Bacillus Subtilis ◽  
Metal Binding ◽  
Metal Ion ◽  
Complex Nature ◽  
Binding Motif ◽  
Copper Chaperone ◽  
Ion Transfer ◽  
Copper Binding ◽  
Binding Properties ◽  
In The Wild

Understanding the metal-binding properties and solution states of metallo-chaperones is a key step in understanding how they function in metal ion transfer. Using spectroscopic, bioanalytical and biochemical methods, we have investigated the copper-binding properties and association states of the putative copper chaperone of Bacillus subtilis, CopZ, and a variant of the protein lacking the two cysteine residues of the MXCXXC copper-binding motif. We show that copper-free CopZ exists as a monomer, but that addition of copper(I) causes the protein to associate into homodimers. The nature of the copper(I)—CopZ complex is dependent on the level of copper loading, and we report the detection of three distinct forms, containing 0.5, 1.0 and 1.5 copper(I) ions per protein. The presence of excess dithiothreitol has a significant effect on copper(I) binding to CopZ, such that, in its presence, copper(I)—CopZ occurs mainly as a monomer species. Data for copper binding to the double-cysteine variant of CopZ are consistent with an essential role for these residues in tight copper binding in the wild-type protein. We conclude that the complex nature of copper(I) binding to CopZ may underpin mechanisms of protein-to-protein copper(I) transfer.

scholarly journals The bacterial copper resistance protein CopG contains a cysteine-bridged tetranuclear copper cluster

2020 ◽  
Vol 295 (32) ◽  
pp. 11364-11376 ◽  
Author(s):  
Andrew C. Hausrath ◽  
Nicholas A. Ramirez ◽  
Alan T. Ly ◽  
Megan M. McEvoy
Keyword(s):  
Metal Binding ◽  
Biochemical Characterization ◽  
Copper Ions ◽  
Three Dimensional ◽  
Copper Resistance ◽  
Dimensional Structure ◽  
Copper Binding ◽  
Uncharacterized Protein ◽  
X Ray Crystallography ◽  
Biochemical Analyses

CopG is an uncharacterized protein ubiquitous in Gram-negative bacteria whose gene frequently occurs in clusters of copper resistance genes and can be recognized by the presence of a conserved CxCC motif. To investigate its contribution to copper resistance, here we undertook a structural and biochemical characterization of the CopG protein from Pseudomonas aeruginosa. Results from biochemical analyses of CopG purified under aerobic conditions indicate that it is a green copper-binding protein that displays absorbance maxima near 411, 581, and 721 nm and is monomeric in solution. Determination of the three-dimensional structure by X-ray crystallography revealed that CopG consists of a thioredoxin domain with a C-terminal extension that contributes to metal binding. We noted that adjacent to the CxCC motif is a cluster of four copper ions bridged by cysteine sulfur atoms. Structures of CopG in two oxidation states support the assignment of this protein as an oxidoreductase. On the basis of these structural and spectroscopic findings and also genetic evidence, we propose that CopG has a role in interconverting Cu(I) and Cu(II) to minimize toxic effects and facilitate export by the Cus RND transporter efflux system.

scholarly journals Hyaluronate/black phosphorus complexes as a copper chelating agent for Wilson disease treatment

2021 ◽  
Vol 25 (1) ◽  
Author(s):  
Seong-Jong Kim ◽  
Hye Hyeon Han ◽  
Sei Kwang Hahn
Keyword(s):  
Targeted Delivery ◽  
Wilson Disease ◽  
Copper Ions ◽  
Binding Capacity ◽  
Genetic Disorder ◽  
Chelating Agent ◽  
Black Phosphorus ◽  
The Body ◽  
Copper Binding

Abstract Background Wilson disease (WD) is a genetic disorder of copper storage, resulting in pathological accumulation of copper in the body. Because symptoms are generally related to the liver, chelating agents capable of capturing excess copper ions after targeted delivery to the liver are highly required for the treatment of WD. Methods We developed hyaluronate-diaminohexane/black phosphorus (HA-DAH/BP) complexes for capturing copper ions accumulated in the liver for the treatment of WD. Results HA-DAH/BP complexes showed high hepatocyte-specific targeting efficiency, selective copper capturing capacity, excellent biocompatibility, and biodegradability. HA enhanced the stability of BP nanosheets and increased copper binding capacity. In vitro cellular uptake and competitive binding tests verified targeted delivery of HA-DAH/BP complexes to liver cells via HA receptor mediated endocytosis. The cell viability test confirmed the high biocompatibility of HA-DAH/BP complexes. Conclusion HA-DAH/BP complexes would be an efficient copper chelating agent to remove accumulated copper in the liver for the WD treatment.

scholarly journals Endo-fucoidan hydrolases from glycoside hydrolase family 107 (GH107) display structural and mechanistic similarities to α-l-fucosidases from GH29

2018 ◽  
Vol 293 (47) ◽  
pp. 18296-18308 ◽  
Author(s):  
Chelsea Vickers ◽  
Feng Liu ◽  
Kento Abe ◽  
Orly Salama-Alber ◽  
Meredith Jenkins ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Biological Activities ◽  
Bacterial Species ◽  
Structural Data ◽  
Side Chain ◽  
Glycoside Hydrolase Family ◽  
X Ray Crystallography ◽  
Catalytic Mechanisms ◽  
Thickening Agents ◽  
Hydrolase Family

Fucoidans are chemically complex and highly heterogeneous sulfated marine fucans from brown macro algae. Possessing a variety of physicochemical and biological activities, fucoidans are used as gelling and thickening agents in the food industry and have anticoagulant, antiviral, antitumor, antibacterial, and immune activities. Although fucoidan-depolymerizing enzymes have been identified, the molecular basis of their activity on these chemically complex polysaccharides remains largely uninvestigated. In this study, we focused on three glycoside hydrolase family 107 (GH107) enzymes: MfFcnA and two newly identified members, P5AFcnA and P19DFcnA, from a bacterial species of the genus Psychromonas. Using carbohydrate-PAGE, we show that P5AFcnA and P19DFcnA are active on fucoidans that differ from those depolymerized by MfFcnA, revealing differential substrate specificity within the GH107 family. Using a combination of X-ray crystallography and NMR analyses, we further show that GH107 family enzymes share features of their structures and catalytic mechanisms with GH29 α-l-fucosidases. However, we found that GH107 enzymes have the distinction of utilizing a histidine side chain as the proposed acid/base catalyst in its retaining mechanism. Further interpretation of the structural data indicated that the active-site architectures within this family are highly variable, likely reflecting the specificity of GH107 enzymes for different fucoidan substructures. Together, these findings begin to illuminate the molecular details underpinning the biological processing of fucoidans.

scholarly journals Effects of the Chromosome Partitioning Protein Spo0J (ParB) on oriC Positioning and Replication Initiation in Bacillus subtilis

2003 ◽  
Vol 185 (4) ◽  
pp. 1326-1337 ◽  
Author(s):  
Philina S. Lee ◽  
Daniel Chi-Hong Lin ◽  
Shigeki Moriya ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Binding Sites ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Significant Proportion ◽  
Subcellular Location ◽  
Replication Initiation ◽  
Null Mutant ◽  
Protein Ratio ◽  
Chromosome Partitioning

ABSTRACT Spo0J (ParB) of Bacillus subtilis is a DNA-binding protein that belongs to a conserved family of proteins required for efficient plasmid and chromosome partitioning in many bacterial species. We found that Spo0J contributes to the positioning of the chromosomal oriC region, but probably not by recruiting the origin regions to specific subcellular locations. In wild-type cells during exponential growth, duplicated origin regions were generally positioned around the cell quarters. In a spo0J null mutant, sister origin regions were often closer together, nearer to midcell. We found, by using a Spo0J-green fluorescent protein [GFP] fusion, that the subcellular location of Spo0J was a consequence of the chromosomal positions of the Spo0J binding sites. When an array of binding sites (parS sites) were inserted at various chromosomal locations in the absence of six of the eight known parS sites, Spo0J-GFP was no longer found predominantly at the cell quarters, indicating that Spo0J is not sufficient to recruit chromosomal parS sites to the cell quarters. spo0J also affected chromosome positioning during sporulation. A spo0J null mutant showed an increase in the number of cells with some origin-distal regions located in the forespore. In addition, a spo0J null mutation caused an increase in the number of foci per cell of LacI-GFP bound to arrays of lac operators inserted in various positions in the chromosome, including the origin region, an increase in the DNA-protein ratio, and an increase in origins per cell, as determined by flow cytometry. These results indicate that the spo0J mutant produced a significant proportion of cells with increased chromosome content, probably due to increased and asynchronous initiation of DNA replication.

scholarly journals The YcnI protein from Bacillus subtilis contains a copper-binding domain

2021 ◽  
pp. 101078
Author(s):  
Madhura S. Damle ◽  
Aarshi N. Singh ◽  
Stephen C. Peters ◽  
Veronika A. Szalai ◽  
Oriana S. Fisher
Keyword(s):  
Bacillus Subtilis ◽  
Binding Domain ◽  
Copper Binding

Copper chaperone Atox1 interacts with the metal-binding domain of Wilson's disease protein in cisplatin detoxification

2013 ◽  
Vol 454 (1) ◽  
pp. 147-156 ◽  
Author(s):  
Nataliya V. Dolgova ◽  
Sergiy Nokhrin ◽  
Corey H. Yu ◽  
Graham N. George ◽  
Oleg Y. Dmitriev
Keyword(s):  
Metal Binding ◽  
Wilson’S Disease ◽  
Wilson's Disease ◽  
Binding Domain ◽  
Binding Motif ◽  
Copper Chaperone ◽  
Copper Binding ◽  
Copper Transporters ◽  
Metal Binding Domain ◽  
Disease Protein

Human copper transporters ATP7B (Wilson's disease protein) and ATP7A (Menkes' disease protein) have been implicated in tumour resistance to cisplatin, a widely used anticancer drug. Cisplatin binds to the copper-binding sites in the N-terminal domain of ATP7B, and this binding may be an essential step of cisplatin detoxification involving copper ATPases. In the present study, we demonstrate that cisplatin and a related platinum drug carboplatin produce the same adduct following reaction with MBD2 [metal-binding domain (repeat) 2], where platinum is bound to the side chains of the cysteine residues in the CxxC copper-binding motif. This suggests the same mechanism for detoxification of both drugs by ATP7B. Platinum can also be transferred to MBD2 from copper chaperone Atox1, which was shown previously to bind cisplatin. Binding of the free cisplatin and reaction with the cisplatin-loaded Atox1 produce the same protein-bound platinum intermediate. Transfer of platinum along the copper-transport pathways in the cell may serve as a mechanism of drug delivery to its target in the cell nucleus, and explain tumour-cell resistance to cisplatin associated with the overexpression of copper transporters ATP7B and ATP7A.

scholarly journals Metal-binding mechanism of Cox17, a copper chaperone for cytochrome c oxidase

2004 ◽  
Vol 382 (1) ◽  
pp. 307-314 ◽  
Author(s):  
Peep PALUMAA ◽  
Liina KANGUR ◽  
Anastassia VORONOVA ◽  
Rannar SILLARD
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Metal Binding ◽  
Copper Ions ◽  
Copper Chaperone ◽  
Copper Chaperones ◽  
Rate Limiting Step ◽  
Charge State Distributions ◽  
Rate Limiting ◽  
Partner Proteins

Cox17, a copper chaperone for cytochrome c oxidase, is an essential and highly conserved protein. The structure and mechanism of functioning of Cox17 are unknown, and even its metalbinding stoichiometry is elusive. In the present study, we demonstrate, using electrospray ionization–MS, that porcine Cox17 binds co-operatively four Cu+ ions. Cu4Cox17 is stable at pH values above 3 and fluorescence spectra indicate the presence of a solvent-shielded multinuclear Cu(I) cluster. Combining our results with earlier EXAFS results on yeast CuCox17, we suggest that Cu4Cox17 contains a Cu4S6-type cluster. At supramillimolar concentrations, dithiothreitol extracts metals from Cu4Cox17, and an apparent copper dissociation constant KCu=13 fM was calculated from these results. Charge-state distributions of different Cox17 forms suggest that binding of the first Cu+ ion to Cox17 causes a conformational change from an open to a compact state, which may be the rate-limiting step in the formation of Cu4Cox17. Cox17 binds non-co-operatively two Zn2+ ions, but does not bind Ag+ ions, which highlights its extremely high metal-binding specificity. We further demonstrate that porcine Cox17 can also exist in partly oxidized (two disulphide bridges) and fully oxidized (three disulphide bridges) forms. Partly oxidized Cox17 can bind one Cu+ or Zn2+ ion, whereas fully oxidized Cox17 does not bind metals. The metal-binding properties of Cox17 imply that, in contrast with other copper chaperones, Cox17 is designed for the simultaneous transfer of up to four copper ions to partner proteins. Metals can be released from Cox17 by non-oxidative as well as oxidative mechanisms.

Toxicity of the First Ten MEIC Chemicals to Bacteria

1993 ◽  
Vol 21 (2) ◽  
pp. 151-155
Author(s):  
Gustaw Kerszman
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Linear Regression ◽  
Minimal Inhibitory Concentration ◽  
Blood Concentration ◽  
Inhibitory Concentration ◽  
Human Lymphocytes ◽  
Bacterial Species ◽  
E Coli ◽  
Mild Toxicity

The toxicity of the first ten MEIC chemicals to Escherichia coli and Bacillus subtilis was examined. Nine of the chemicals were toxic to the bacteria, with the minimal inhibitory concentration (MIC) ranging from 10-3 to 4.4M. The sensitivities of both organisms were similar, but the effect on E. coli was often bactericidal, while it was bacteriostatic for B. subtilis. Digoxin was not detectably toxic to either bacterial species. Amitriptyline and FeSO4 were relatively less toxic to the bacteria than to human cells. For seven chemicals, a highly significant linear regression was established between log MIC in bacteria and log of blood concentration, giving lethal and moderate/mild toxicity in humans, as well as with toxicity to human lymphocytes.

Tetramer formation of Bacillus subtilis YabJ protein that belongs to YjgF/YER057c/UK114 family

2021 ◽  
Vol 85 (2) ◽  
pp. 297-306
Author(s):  
Zui Fujimoto ◽  
Le Thi Thu Hong ◽  
Naomi Kishine ◽  
Nobuhiro Suzuki ◽  
Keitarou Kimura
Keyword(s):  
Bacillus Subtilis ◽  
Quaternary Structure ◽  
Single Amino Acid ◽  
Wild Type ◽  
Amino Acid Mutation ◽  
X Ray Crystallography ◽  
Ligand Binding Sites ◽  
Potential Ligand ◽  
Single Amino Acid Mutation

ABSTRACT Bacillus subtilis YabJ protein belongs to the highly conserved YjgF/YER057c/UK114 family, which has a homotrimeric quaternary structure. The dominant allele of yabJ gene that is caused by a single amino acid mutation of Ser103Phe enables poly-γ-glutamic acid (γPGA) production of B. subtilis under conditions where the cell-density signal transduction was disturbed by the loss of DegQ function. X-ray crystallography of recombinant proteins revealed that unlike the homotrimeric wild-type YabJ, the mutant YabJ(Ser103Phe) had a homotetrameric quaternary structure, and the structural change appeared to be triggered by an inversion of the fifth β-strand. The YabJ homotetramer has a hole that is highly accessible, penetrating through the tetramer, and 2 surface concaves as potential ligand-binding sites. Western blot analyses revealed that the conformational change was also induced in vivo by the Ser103Phe mutation.

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