title: Structural and mechanistic insights into an extracytoplasmic copper trafficking pathway in Streptomyces lividans.
abstract: In Streptomyces lividans an extracytoplasmic copper-binding Sco protein plays a role in two unlinked processes: (i) initiating a morphological development switch and (ii) facilitating the co-factoring of the CuA domain of CcO (cytochrome c oxidase). How Sco obtains copper once secreted to the extracytoplasmic environment is unknown. In the present paper we report on a protein possessing an HX₆MX₂₁HXM motif that binds a single cuprous ion with subfemtomolar affinity. High-resolution X-ray structures of this extracytoplasmic copper chaperone-like protein (ECuC) in the apo- and Cu(I)-bound states reveal that the latter possesses a surface-accessible cuprous-ion-binding site located in a dish-shaped region of β-sheet structure. A cuprous ion is transferred under a favourable thermodynamic gradient from ECuC to Sco with no back transfer occurring. The ionization properties of the cysteine residues in the Cys⁸⁶xxxCys⁹⁰ copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys⁸⁶ in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Δsco, Δecuc and Δsco/ecuc, and subsequent in vivo assays lend support to the existence of a branched extracytoplasmic copper-trafficking pathway in S. lividans. One branch requires both Sco and to a certain extent ECuC to cofactor the CuA domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.
authors.affiliation: *School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
authors.forename: Katie L I M
authors.initials: KL
authors.lastname: Blundell
authors.affiliation: *School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hough
authors.affiliation: †Molecular Biotechnology, Institute of Biology, Sylvius Laboratory, Leiden University, PO Box 9505, 2300RA Leiden, The Netherlands.
authors.forename: Erik
authors.initials: E
authors.lastname: Vijgenboom
authors.affiliation: *School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, U.K.
authors.forename: Jonathan A R
authors.initials: JA
authors.lastname: Worrall
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003576:Electron Transport Complex IV / Q000737:chemistry / Q000378:metabolism; D055786:Gene Knockout Techniques; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D056511:Metallochaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D048170:Streptomyces lividans / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D056511:Metallochaperones; D050505:Mutant Proteins; D011994:Recombinant Proteins; C023548:copper-binding protein; D003300:Copper; D003576:Electron Transport Complex IV; D003545:Cysteine