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

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 Cu-A 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 beta-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 Cys(86)xxxCys(90) copper-binding motif of Sco, together with their positional locations identified from an X-ray structure of Sco, suggests a role for Cys(86) in initiating an inter-complex ligand-exchange reaction with Cu(I)-ECuC. Generation of the genetic knockouts, Delta sco, Delta ecuc and Delta sco/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 Cu-A domain, whereas the other uses only Sco to deliver copper to a cuproenzyme to initiate morphological development.