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) Membrane transporters in the cation diffusion facilitator family are found both in eukaryotes and prokaryotes (1). This protein family of more than 400 genetically related members is characterized by a homologous hydrophobic N-terminal domain followed by a hydrophilic C-terminal domain that is variable both in sequence and length (2). Despite sequence variability, all CDF 3 family members exclusively transport zinc and other divalent metal ions across the cytoplasm or organelle membranes, thus playing a variety of important roles in cellular zinc homeostasis controls (3-7 may be an additional substrate (12). Both Zn 2ϩ and Fe 2ϩ are borderline soft metal ions with a similar donor preference for a combination of three protein ligand groups as follows: the cysteine thiolate, histidine imidazole, and aspartate/glutamate carboxylate (16). Phenotype analyses of two homologous CDF transporters, CzcD from Ralstonia metallidurans and ZitB from E. coli, demonstrated that mutating a highly conserved Asp residue in the putative TM-5 rendered host cells hypersensitive to zinc, probably because of a loss of zinc efflux pumping activities (17). The Asp appeared to be essential because expression of CzcD D158E , CzcD D158A , ZitB D163E , and ZitB D163A mutant proteins conferred no zinc resistance. It is not clear, however, whether this conserved CDF aspartate is directly involved in binding and transport of Zn 2ϩ and/or Fe 2ϩ . In the present study, we sought to establish the functional role of the equivalent Asp residue in YiiP (Asp-157) by examining the effects of D157A and D157C mutations on metal binding and transport by using direct biophysical measurements. Furthermore, Asp-157 was localized to the hydrophobic core of TM-5 to establish a structural connection with the substrate translocation pathway where metal ions are selected and transported across the membrane. Metal binding to Asp-157 was found to be highly specific, with a strict selectivity for Zn 2ϩ and Cd 2ϩ over Hg 2ϩ , Fe 2ϩ , and other divalent metal ions that are thought to be the frequent CDF substrates. Because Asp-157 is one of the most conserved residues in the CDF family, selective metal binding to Asp-157 has broad structural and mechanistic implications..
EXPERIMENTAL PROCEDURESSite-directed Mutagenesis-Cloning and construction of the expression plasmid pYiiP-His were described previously (15). Site-directed mutagenesis was performed using the QuickChange site-directed mutagenesis kit (Stratagene). A mutant C287S was first prepared using * This work was supported by the Labora...