Periplasmic domain of CusA in an Escherichia coli Cu⁺/Ag⁺ transporter has metal binding sites

The resistance nodulation division (RND)-type efflux systems are utilized in Gram-negative bacteria to export a variety of substrates. The CusCFBA system is the Cu⁺ and Ag⁺ efflux system in Escherichia coli, conferring resistance to lethal concentrations of Cu⁺ and Ag⁺. The periplasmic component, CusB, which is essential for the assembly of the protein complex, has Cu⁺ or Ag⁺ binding sites. The twelve-span membrane protein CusA is a homotrimeric transporter, and has a relatively large periplasmic domain. Here, we constructed the periplasmic domain of CusA by joining two DNA segments and then successfully expressed and purified the protein. Isothermal titration calorimetry experiments revealed Ag⁺ binding sites with Kds of 10^-6-10^-5 M. Our findings suggest that the metal binding in the periplasmic domain of CusA might play an important role in the function of the efflux pump.