Selective metal binding to a membrane-embedded aspartate in the Escherichia coli metal transporter YiiP (FieF)

The cation diffusion facilitators (CDF) are a ubiquitous family of metal transporters that play important roles in homeostasis of a wide range of divalent metal cations. Molecular identities of substrate-binding sites and their metal selectivity in the CDF family are thus far unknown. By using isothermal titration calorimetry and stopped-flow spectrofluorometry, we directly examined metal binding to a highly conserved aspartate in the Escherichia coli CDF transporter YiiP (FieF). A D157A mutation abolished a Cd²⁺-binding site and impaired the corresponding Cd²⁺ transport. In contrast, substitution of Asp-157 with a cysteinyl coordination residue resulted in intact Cd²⁺ binding as well as full transport activity. A similar correlation was found for Zn²⁺ binding and transport, suggesting that Asp-157 is a metal coordination residue required for binding and transport of Cd²⁺ and Zn²⁺. The location of Asp-157 was mapped topologically to the hydrophobic core of transmembrane segment 5 (TM-5) where D157C was found partially accessible to thiol-specific labeling of maleimide polyethylene-oxide biotin. Binding of Zn²⁺ and Cd²⁺, but not Fe²⁺, Hg²⁺, Co²⁺, Ni²⁺, Mn²⁺, Ca²⁺, and Mg²⁺, protected D157C from maleimide polyethylene-oxide biotin labeling in a concentration-dependent manner. Furthermore, isothermal titration calorimetry analysis of YiiP_D157A showed no detectable change in Fe²⁺ and Hg²⁺ calorimetric titrations, indicating that Asp-157 is not a coordination residue for Fe²⁺ and Hg²⁺ binding. Our results provided direct evidence for selective binding of Zn²⁺ and Cd²⁺ to the highly conserved Asp-157 and defined its functional role in metal transport.