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J. Biol. Chem., Vol. 283, Issue 17, 11721-11733, April 25, 2008

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Probing Structure-Function Relationships and Gating Mechanisms in the CorA Mg²⁺ Transport System^*

Jian Payandeh¹, Canhui Li^¶||**, Mohabir Ramjeesingh^¶**, Ewa Poduch, Christine E. Bear^¶||**, and Emil F. Pai^¶2

From the Departments of Medical Biophysics, ^¶Biochemistry, and Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1L7, Canada, Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, Toronto, Ontario M5G 1L7, Canada, ^||Molecular Structure and Function Programme, ^**Research Institute in the Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada, and Center for Molecular Design and Preformulations, Toronto General Research Institute, Toronto, Ontario M5G 1L7, Canada

Recent crystal structures of the CorA Mg²⁺ transport protein from Thermotoga maritima (TmCorA) revealed an unusually long ion pore putatively gated by hydrophobic residues near the intracellular end and by universally conserved asparagine residues at the periplasmic entrance. A conformational change observed in an isolated funnel domain structure also led to a proposal for the structural basis of gating. Because understanding the molecular mechanisms underlying ion channel and transporter gating remains an important challenge, we have undertaken a structure-guided engineering approach to probe structure-function relationships in TmCorA. The intracellular funnel domain is shown to constitute an allosteric regulatory module that can be engineered to promote an activated or closed state. A periplasmic gate centered about a proline-induced kink of the pore-lining helix is described where "helix-straightening" mutations produce a dramatic gain-of-function. Mutation to the narrowest constriction along the pore demonstrates that a hydrophobic gate is operational within this Mg²⁺-selective transport protein and likely forms an energetic barrier to ion flux. We also provide evidence that highly conserved acidic residues found in the short periplasmic loop are not essential for TmCorA function or Mg²⁺ selectivity but may be required for proper protein folding and stability. This work extends our gating model for the CorA-Alr1-Mrs2 superfamily and reveals features that are characteristic of an ion channel. Aspects of these results that have broader implications for a range of channel and transporter families are highlighted.

Received for publication, September 20, 2007 , and in revised form, January 30, 2008.

^* This study was funded by the Canada Research Chairs Program, the National Sciences and Engineering Research Council of Canada, the Canadian Institutes of Health Research (to C. E. B. and E. F. P.), and the Ontario Research and Development Challenge Fund (to E. F. P.). 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. 1-6.

¹ To whom correspondence may be addressed: Div. of Cancer Genomics & Proteomics, Ontario Cancer Inst., Medical and Related Sciences Centre, 101 College St., Toronto, Ontario M5G 1L7, Canada. Tel.: 416-581-7545; Fax: 416-581-7545; E-mail: payandeh{at}uhnres.utoronto.ca.

² To whom correspondence may be addressed. Tel.: 416-581-7545; Fax: 416-581-7545; E-mail: pai{at}hera.med.utoronto.ca.