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

Abstract

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. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.