Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in <i>Streptomyces lividans</i>

Blundell, Katie L. I. M.; Hough, Michael A.; Vijgenboom, Erik; Worrall, J.A.R.

doi:10.1042/bj20140017

Cited by 23 publications

(37 citation statements)

References 54 publications

(84 reference statements)

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“…58 The copD gene is followed by a gene encoding a putative member of the DUF461 family, which includes the proteins known as PCu A C, PcuC, and EcuC. 35,59–61 This entire gene cluster forms an operon that is coregulated in response to copper. 37 The predicted Mst -CopC protein includes an N-terminal signal peptide for export to the periplasm.…”

Section: Resultsmentioning

confidence: 99%

“…16,33,34 Finally, operons containing copC and copD without copA , copB , or copE have been observed in multiple organisms. 18,33,35 For example, in methanotrophic bacteria, copC and copD genes are located adjacent to the operon encoding particulate methane monooxygenase (pMMO), a copper-containing enzyme that catalyzes the first step of methane metabolism. 36 Notably, the copD gene in a mutant strain of Methylosinus trichosporium OB3b that is impaired in cytoplasmic copper uptake and regulation contains a frameshift deletion, consistent with CopC and CopD functioning in copper import in this organism.…”

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confidence: 99%

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The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

et al. 2016

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The CopC proteins are periplasmic copper binding proteins believed to play a role in bacterial copper homeostasis. Previous studies have focused on CopCs that are part of seven-protein Cop or Pco systems involved in copper resistance. These canonical CopCs contain distinct Cu(I) and Cu(II) binding sites. Mounting evidence suggests that CopCs are more widely distributed, often present only with the CopD inner membrane protein, frequently as a fusion protein, and that the CopC and CopD proteins together function in the uptake of copper to the cytoplasm. In the methanotroph Methylosinus trichosporium OB3b, genes encoding a CopCD pair are located adjacent to the particulate methane monooxygenase (pMMO) operon. The CopC from this organism (Mst-CopC) was expressed, purified, and structurally characterized. The 1.46 Å resolution crystal structure of Mst-CopC reveals a single Cu(II) binding site with coordination somewhat different from that in canonical CopCs, and the absence of a Cu(I) binding site. Extensive bioinformatic analyses indicate that the majority of CopCs in fact contain only a Cu(II) site, with just 10% of sequences corresponding to the canonical two-site CopC. Accordingly, a new classification scheme for CopCs was developed, and detailed analyses of the sequences and their genomic neighborhoods reveal new proteins potentially involved in copper homeostasis, providing a framework for expanded models of CopCD function.

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Section: Resultsmentioning

confidence: 99%

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The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

et al. 2016

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“…Under Cu stress conditions, Cu + ions bind to the DNA bound apo-CsoR and allosterically activate transcriptional de-repression of efflux systems involving multiple P1-type ATPases, and CopZ-like metallochaperones to rapidly remove Cu + from the cytosol 16,17 . Under Cu limiting conditions (homeostasis) an extracytoplasmic Cu pathway involving two Cu metallochaperones (Sco and ECuC) is operable 8,19 . This pathway serves to metallate the dinuclear CuA site in the aa3-type cytochrome c oxidase (CcO) 8 and the mononuclear Cu site in the Cu-radical oxidase GlxA 9,10 .…”

Section: Introductionmentioning

confidence: 99%

A cytosolic copper storage protein provides a second level of copper tolerance inStreptomyces lividans

et al. 2018

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Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P-type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

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“…Recent studies in S. lividans have identified an extracellular copper-trafficking pathway involving two copper chaperone proteins (Sco and ECuC) (34,35) and a DyP-type heme peroxidase (DtpA) (36) that are all required to facilitate GlxA maturation (i.e. copper-loading and subsequent Tyr-Cys cross-link formation (30)).…”

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Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

Chaplin

Wilson

Hough

et al. 2016

Journal of Biological Chemistry

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Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.Lytic polysaccharide monooxygenases (LPMOs) 3 are enzymes that utilize copper as their functional active site metal and act to enhance the depolymerization of recalcitrant polysaccharides in nature such as cellulose and chitin (1, 2). Identification and characterization of members of this cuproenzyme family from fungal and bacterial species have gathered pace in recent years due in part to their promising uses in biorefinery applications of biomass to derive second generation biofuels (2, 3). LPMOs are classed into auxiliary activity (AA) families in the CAZy (carbohydrate active enzyme) database with four AA families, AA9, AA10, AA11, and AA13 identified so far (4). AA9 (formerly GH61) and AA10 (formerly CBM33) families have been the most extensively studied, with AA9 members having activity for cellulose, hemicellulose, and cellodextrin substrates (5-8) and AA10 members having activity with either cellulose or chitin substrates (9, 10). The AA11 and AA13 families are the most recently discovered, and so far have been determined to have activity with chitin and starch substrates, respectively (11, 12). All LPMOs coordinate a single copper ion by three nitrogen ligands to from the active site. Two of these are provided from the amino group (Nt) and side chain of the N-terminal His with the third from a second His side chain to giv...

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Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans

Cited by 23 publications

References 54 publications

The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

The CopC Family: Structural and Bioinformatic Insights into a Diverse Group of Periplasmic Copper Binding Proteins

A cytosolic copper storage protein provides a second level of copper tolerance inStreptomyces lividans

Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases

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