Binding and transport of metal ions at the dimer interface of the Escherichia coli metal transporter YiiP

YiiP is a representative member of the cation diffusion facilitator (CDF) family, a class of ubiquitous metal transporters that play an essential role in metal homeostasis. Recently, a pair of Zn ²⁺/Cd ²⁺-selective binding sites has been localized to two highly conserved aspartyl residues (Asp ¹⁵⁷), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp ¹⁵⁷ and yet another highly conserved Asp ⁴⁹ in the TM2. Calorimetric binding analysis indicated that Asp ⁴⁹ and Asp ¹⁵⁷ contribute to a common Cd ²⁺ binding site in each subunit. Copper phenanthroline oxidation of YiiP _D49C, YiiP _D157C, and YiiP _D49C/D157C yielded inter- and intra-subunit cross-links among Cys ⁴⁹ and Cys ¹⁵⁷, consistent with the spatial proximity of two (Asp ⁴⁹-Asp ¹⁵⁷) sites at the dimer interface. Hg ²⁺ binding to YiiP _D49C or YiiP _D49C/D157C also yielded a Cys ⁴⁹-Hg ²⁺-Cys ⁴⁹ biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp ⁴⁹-Asp ¹⁵⁷) ₂ bimetal binding center. Two bound Cd ²⁺ ions were found transported cooperatively with a sigmoidal dependence on the Cd ²⁺ concentration (n = 1.4). The binding affinity, transport cooperativity, and rate were modestly reduced by either a D49C or D157C mutation, but greatly diminished when all the bidentate aspartate O-ligands in (Asp ⁴⁹-Asp ¹⁵⁷) ₂ were replaced by the monodentate cysteine S-ligands. The functional significance of these findings is discussed based on the unique coordination chemistry of aspartyl residues and a model for the translocation pathway of metal ions at the YiiP dimer interface.