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 Zn2+/Cd2+-selective binding sites has been localized to two highly conserved aspartyl residues (Asp157), each in a 2-fold-symmetry-related transmembrane segment 5 (TM5) of a YiiP homodimer. Here we report the functional and structural interactions between Asp157 and yet another highly conserved Asp49 in the TM2. Calorimetric binding analysis indicated that Asp49 and Asp157 contribute to a common Cd2+ 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 Cys49 and Cys157, consistent with the spatial proximity of two (Asp49-Asp157) sites at the dimer interface. Hg2+ binding to YiiP(D49C) or YiiP(D49C/D157C) also yielded a Cys49-Hg2+-Cys49 biscysteinate complex across the dimer interface, further establishing the interfacial location of a (Asp49-Asp157)2 bimetal binding center. Two bound Cd2+ ions were found transported cooperatively with a sigmoidal dependence on the Cd2+ 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 (Asp49-Asp157)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.
Highlights
cation diffusion facilitator (CDF) is a ubiquitous family of metal transporters found in prokaryotes and eukaryotes (9)
Dimer Interface—Aspartyl residues are frequently found in metal binding clusters where they bridge a pair of metal ions by providing two oxygen ligands, each interacting with one of the two adjacent metal ions (14)
The experiments described establish a strong correlation between YiiP transport and metal binding to two highly conserved aspartyl residues (Asp[49] and Asp157)
Summary
We explored how protein tertiary folding and subunit assembly may contribute to the Zn2ϩ/Cd2ϩ translocation pathway in YiiP. Toward this end, we first characterized the functional role of Asp[49] by examining the effects of D49A and D49C mutations on metal binding and transport. Our results suggest that the four highly conserved aspartyl residues form a (Asp49-Asp157)[2] binding center for binding and cooperative transport of two Cd2ϩ ions at the dimer interface. Further mutation-function characterization identified two neighboring residues, Asp[45] and His[153], contributing to the bimetal binding center. Our experiments led to a model for the translocation pathway of metal ions at the YiiP dimer interface
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