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+ for to the highly conserved Asp-157 and defined its functional role in metal transport.