title: CopC protein from Pseudomonas fluorescens SBW25 features a conserved novel high-affinity Cu(II) binding site.
abstract: Copper homeostasis in the bacterium Pseudomonas fluorescens SBW25 appears to be mediated mainly via chromosomal cue and cop systems. Under elevated copper levels that induce stress, the cue system is activated for expression of a P1B-type ATPase to remove excess copper from the cytosol. Under copper-limiting conditions, the cop system is activated to express two copper uptake proteins, Pf-CopCD, to import this essential nutrient. Pf-CopC is a periplasmic copper chaperone that may donate copper to the inner membrane transporter Pf-CopD for active copper importation. A database search revealed that Pf-CopC belongs to a new family of CopC proteins (designated Type B in this work) that differs significantly from the known CopC proteins of Type A that possess two separated binding sites specific for Cu(I) and Cu(II). This article reports the isolation and characterization of Pf-CopC and demonstrates that it lacks a Cu(I) binding site and possesses a novel Cu(II) site that binds Cu(II) with 100 times stronger affinity than do the Type A proteins. Presumably, this is a requirement for a copper uptake role under copper-limiting conditions. The Cu(II) site incorporates a highly conserved amino terminal copper and nickel (ATCUN) binding motif, NH2-Xxx-Xxx-His, but the anticipated ATCUN binding mode is prevented by a thermodynamically more favorable binding mode comprising His1 as a key bidentate ligand and His3 and His85 as co-ligands. However, upon His1 mutation, the ATCUN binding mode is adopted. This work demonstrates how a copper chaperone may fine tune its copper binding site to meet new challenges to its function.
authors.affiliation: School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne , Parkville, Victoria 3010, Australia.
authors.forename: Chathuri J K
authors.initials: CJ
authors.lastname: Wijekoon
authors.forename: Tessa R
authors.initials: TR
authors.lastname: Young
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Wedd
authors.forename: Zhiguang
authors.initials: Z
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D017124:Conserved Sequence; D003300:Copper / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D011551:Pseudomonas fluorescens; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; C071265:CopC protein, Bacteria; D003300:Copper