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J. Biol. Chem., Vol. 280, Issue 40, 33716-33724, October 7, 2005

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Selective Metal Binding to a Membrane-embedded Aspartate in the Escherichia coli Metal Transporter YiiP (FieF)^*

From the Department of Biology, Brookhaven National Laboratory, Upton, New York 11973

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.

Received for publication, June 3, 2005 , and in revised form, July 26, 2005.

^* This work was supported by the Laboratory Directed Research and Development Program of the Brookhaven National Laboratory, Project 05-064 (to Y. W. and D. F.), and by a National Institutes of Health Grant RO1 GM65137 (to D. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Goldhabor postdoctoral fellow.

² To whom correspondence should be addressed: Biology Dept., Bldg. 463, Brookhaven National Laboratory, Upton, NY 11973. Tel.: 631-344-4208; Fax: 631-344-3407: E-mail: dax{at}bnl.gov.

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