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J. Biol. Chem., Vol. 281, Issue 37, 26813-26820, September 15, 2006

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Escherichia coli CorA Periplasmic Domain Functions as a Homotetramer to Bind Substrate^*

From the Department of Biological Sciences and Biotechnology, State-Key Lab of Biomembranes and Membrane Biotechnology, Tsinghua University, Beijing 100084, China

CorA is a primary Mg²⁺ transporter in bacteria, which also mediates influx of Ni²⁺ and Co²⁺. Topological studies suggested that it could be divided into a large soluble periplasmic domain (PPD) and three membrane-spanning -helixes. In the present study, glutathione S-transferase (GST) fusion Escherichia coli CorA PPD was purified by GST affinity chromatography, and PPD was obtained by on-column thrombin digestion. Size-exclusion chromatography indicated that purified PPD exists as a homotetramer. Single particle electron microscopy analysis of PPD and two-dimensional crystals of GST-PPD indicated that E. coli CorA PPD is a pyramid-like homotetramer with a central cavity. Comparison of the CD spectra of full-length CorA and PPD also suggested that PPD has similar secondary structure to the full-length CorA. Dissociation constants for CorA and PPD with their substrates, determined by dose-dependent fluorescence quench of ligands, suggested that purified PPD retains its substrate binding ability as native CorA. The CorA PPD structure described here may provide structural information for the E. coli CorA functional oligomeric state.

Received for publication, March 13, 2006 , and in revised form, July 10, 2006.

^* This work was supported by the National Nature Science Foundation of China (Grants 30340420442 and 30330160) and the National Basic Research Program of China (Grant 2004CB720005). 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 additional text, references, Table S1, and Figs. S1 and S2.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed. Tel.: 86-10-6278-4768; Fax: 86-10-6279-3367; E-mail: suisf{at}mail.tsinghua.edu.cn.

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