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J. Biol. Chem., Vol. 283, Issue 22, 15142-15151, May 30, 2008

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A Histidine-rich and Cysteine-rich Metal-binding Domain at the C Terminus of Heat Shock Protein A from Helicobacter pylori

IMPLICATION FOR NICKEL HOMEOSTASIS AND BISMUTH SUSCEPTIBILITY^*

From the Department of Chemistry and the Open Laboratory of Chemical Biology, The University of Hong Kong, Pokfulam Road, Hong Kong, China

HspA, a member of the GroES chaperonin family, is a small protein found in Helicobacter pylori with a unique histidine- and cysteine-rich domain at the C terminus. In this work, we overexpressed, purified, and characterized this protein both in vitro and in vivo. The apo form of the protein binds 2.10 ± 0.07 Ni²⁺ or 1.98 ± 0.08 Bi³⁺ ions/monomer with a dissociation constant (K_d) of 1.1 or 5.9 x 10^-19 µM, respectively. Importantly, Ni²⁺ can reversibly bind to the protein, as the bound nickel can be released either in the presence of a chelating ligand, e.g. EDTA, or at an acidic pH (pH 3.8 ± 0.2). In contrast, Bi³⁺ binds almost irreversibly to the protein. Both gel filtration chromatography and native electrophoresis demonstrated that apo-HspA exists as a heptamer in solution. Unexpectedly, binding of Bi³⁺ to the protein altered its quaternary structure from a heptamer to a dimer, indicating that bismuth may interfere with the biological functions of HspA. When cultured in Ni²⁺-supplemented M9 minimal medium, Escherichia coli BL21(DE3) cells expressing wild-type HspA or the C-terminal deletion mutant clearly indicated that the C terminus might protect cells from high concentrations of external Ni²⁺. However, an opposite phenomenon was observed when the same E. coli hosts were grown in Bi³⁺-supplemented medium. HspA may therefore play a dual role: to facilitate nickel acquisition by donating Ni²⁺ to appropriate proteins in a nickel-deficient environment and to carry out detoxification via sequestration of excess nickel. Meanwhile, HspA can be a potential target of the bismuth antiulcer drug against H. pylori.

Received for publication, January 23, 2008 , and in revised form, March 17, 2008.

^* This work was supported in part by Grants HKU7039/04P and HKU7042/07P from the Research Grants Council of Hong Kong, Grants HKUST4/03C and HKU1/07C from the Research Grants Council Central Allocation, the Area of Excellence Scheme of the University Grants Committee, and The University of Hong Kong. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S5.

¹ Supported by a studentship from The University of Hong Kong.

² To whom correspondence should be addressed: Dept. of Chemistry, The University of Hong Kong, Pokfulam Rd., Hong Kong, China. Tel.: 852-2859-8974; Fax: 852-2857-1586; E-mail: hsun{at}hku.hk.

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