title: Heterologous production and functional and thermodynamic characterization of cation diffusion facilitator (CDF) transporters of mesophilic and hyperthermophilic origin.
abstract: The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 μm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+ transport across membranes.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.
authors.forename: Devrishi
authors.initials: D
authors.lastname: Goswami
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
authors.forename: Sachin
authors.initials: S
authors.lastname: Surade
authors.forename: Ernst
authors.initials: E
authors.lastname: Grell
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D027682:Cation Transport Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000235:genetics*; D019216:Metals, Heavy / Q000378:metabolism; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012486:Salmonella typhimurium / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D019216:Metals, Heavy