Heterologous production and functional and thermodynamic characterization of cation diffusion facilitator (CDF) transporters of mesophilic and hyperthermophilic origin

• Published in: Biological chemistry Vol. 393; no. 7; pp. 617 - 629
• Main Authors: Goswami, Devrishi, Kaur, Jagdeep, Surade, Sachin, Grell, Ernst, Michel, Hartmut
• Format: Journal Article
• Language: English
• Published: Germany Walter de Gruyter 01.07.2012
• Subjects: Amino Acid Sequence; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Cation Transport Proteins - biosynthesis; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cloning, Molecular; Computational Biology; Escherichia coli - cytology; Escherichia coli - genetics; functional complementation; Gram-Negative Bacteria - genetics; heavy metal ion coordination; isothermal titration calorimetry; membrane protein; Metals, Heavy - metabolism; Molecular Sequence Data; oligomerization studies; Protein Multimerization; Protein Structure, Quaternary; Salmonella typhimurium - genetics; Thermodynamics; zinc homeostasis
• DOI: 10.1515/hsz-2012-0101
• ISSN: 1431-6730

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 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 and from the hyperthermophilic bacterium , in host cells. STM0758 of 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 was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd and two Zn per protein monomer with K values of around 100 n and 1 μ , respectively, was observed. In addition, at least one more Zn can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn transport across membranes.