Uncovering the Transmembrane Metal Binding Site of the Novel Bacterial Major Facilitator Superfamily-Type Copper Importer CcoA

Abstract

Uptake and trafficking of metals and their delivery to their respective metalloproteins are important processes. Cells need precise control of each step to avoid exposure to excessive metal concentrations and their harmful consequences. Copper (Cu) is a required micronutrient used as a cofactor in proteins. However, in large amounts, it can induce oxidative damage; hence, Cu homeostasis is indispensable for cell survival. Biogenesis of respiratory heme-Cu oxygen (HCO) reductases includes insertion of Cu into their catalytic subunits to form heme-Cu binuclear centers. Previously, we had shown that CcoA is a major facilitator superfamily (MFS)-type bacterial Cu importer required for biogenesis of cbb3-type cytochrome c oxidase (cbb3-Cox). Here, using Rhodobacter capsulatus, we focused on the import and delivery of Cu to cbb3-Cox. By comparing the CcoA amino acid sequence with its homologues from other bacterial species, we located several well-conserved Met, His, and Tyr residues that might be important for Cu transport. We determined the topology of the transmembrane helices that carry these residues to establish that they are membrane embedded, and substituted for them amino acids that do not ligand metal atoms. Characterization of these mutants for their uptake of radioactive (64)Cu and cbb3-Cox activities demonstrated that Met233 and His261 of CcoA are essential and Met237 and Met265 are important, whereas Tyr230 has no role for Cu uptake or cbb3-Cox biogenesis. These findings show for the first time that CcoA-mediated Cu import relies on conserved Met and His residues that could act as metal ligands at the membrane-embedded Cu binding domain of this transporter.

Importance: Cu is a micronutrient that is both essential and toxic; hence, its cellular homeostasis is crucial. Respiratory cbb3-type cytochrome c oxidases (cbb3-Cox) are Cu-containing energy-transducing enzymes that are important for many microaerophilic processes, including photosynthesis, respiration, and bacterial pathogenesis. How Cu is incorporated into cbb3-Cox enzymes is not well known. So far, CcoA is the only known major facilitator superfamily (MFS)-type transporter required for Cu import into the bacterial cytoplasm and for cbb3-Cox biogenesis. This study shows that the membrane-embedded, universally conserved Met and His residues of CcoA are essential for its Cu import function and also for its role in cbb3-Cox biogenesis, shedding light on the mechanism of function of this bacterial prototypical Cu importer.

FIG 1

Sequence alignment of R. capsulatus CcoA homologues and its homology-based 3D structure. (A) Partial alignment of the R. capsulatus CcoA amino acid sequence with its homologues from selected proteobacteria. The conserved Met, His, and Tyr residues are highlighted, and their amino acid numbers in R. capsulatus CcoA are indicated (see Table S1 for the corresponding amino acid positions in the other species). (B) Homology model of R. capsulatus CcoA constructed with Swiss-Model (http://swissmodel.expasy.org) using the E. coli YaJR protein (PDB code 3WDO), which is the closest homologue of R. capsulatus CcoA (16% sequence identity), as the template.

FIG 2

Membrane topology of the TM7, -8, and -9 of R. capsulatus CcoA. (A) The TMRPres2D program predicted topology of the TM7, TM8, and TM9 of CcoA was used to fuse LacZ and PhoA to His249, located between TM7 and TM8, and to Arg281, located between TM8 and TM9. The conserved residues Met233, Met237, His261, and Met265, located in TM7 and TM8, and the PhoA and LacZ phenotypes of the fusions are indicated. (B) Immunoblot analysis of CcoA-PhoA and CcoA-LacZ fusions. Chromatophore membranes from cells carrying no fusion (ΔccoA) or the CcoA-LacZ (CcoA249::LacZ and CcoA281::LacZ) and CcoA-Pho (CcoA249::PhoA and CcoA281::PhoA) protein fusions were probed with polyclonal antibodies against E. coli alkaline phosphatase (anti-PhoA) or β-galactosidase (anti-LacZ), as indicated. The double band seen with the CcoA249::PhoA fusion protein was attributed to proteolytic degradation (38). (C) β-Galactosidase (for the CcoA-LacZ fusions) and alkaline phosphatase (for the CcoA-PhoA fusions) activities of chromatophore membranes of R. capsulatus cells harboring the appropriate fusions.

FIG 3

l-Arabinose inducible production of R. capsulatus CcoA in E. coli and R. capsulatus. (A) R. capsulatus ccoA was first cloned into the expression plasmid pBAD/Myc-His (pBK68) to provide an inducible and C-terminally Myc-His epitope-tagged ccoA construct. The plasmid pBK68 was then integrated into the broad-host-range plasmid pRK415 to yield pBK69, which is conjugally transferable into R. capsulatus. (B) Immunoblot analysis of R. capsulatus CcoA expressed in E. coli and in R. capsulatus. Membranes prepared from l-Ara induced E. coli (LMG194) and R. capsulatus ΔccoA (SE8) strains harboring appropriate plasmids expressing wild-type CcoA or mutant derivatives (as indicated), grown with or without 0.5% l-Ara, and probed with anti-Myc monoclonal antibodies.

FIG 4

Whole-cell uptake of radioactive ⁶⁴Cu by native and mutant variants of CcoA. Uptake kinetics was determined using E. coli LMG194 expressing native, single-mutant (A) and double-mutant (B) derivatives of CcoA, as indicated. The strain LMG194 carrying pBAD/Myc-His without ccoA was used to monitor the CcoA-independent background ⁶⁴Cu uptake in E. coli (A, inset). This CcoA-independent ⁶⁴Cu uptake was subtracted from the data obtained using native and mutant CcoA variants, and corrected values were plotted as a function of time. Each assay was repeated at least three times using independently grown cultures, and for each time point, the measured values were within ±10% of each other.

FIG 5

Proposed transmembrane Cu binding sites of CcoA. A hypothetical model of R. capsulatus CcoA illustrating its putative trigonal (Met₂₃₃, His₂₆₁, and Met₂₆₅) or tetrahedral (with additional Met₂₃₇ or Met₂₂₇) Cu-ligating amino acid residues is shown. For the sake of simplicity, only TM7 and TM8 of CcoA are shown. The Cys247 and His249 residues that are located on the periplasmic loop between TM7 and TM8 and might be involved in early steps of Cu recognition are also shown.