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J. Biol. Chem., Vol. 280, Issue 50, 41716-41724, December 16, 2005

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Structures of the MthK RCK Domain and the Effect of Ca²⁺ on Gating Ring Stability^*

Jianbo Dong1, Ning Shi1, Ian Berke, Liping Chen, and Youxing Jiang2

From the Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390 and the Department of Molecular Medicine, Cornell University, Ithaca, New York 14853

MthK is a Ca²⁺-gated K⁺ channel from Methanobacterium autotrophicum. The crystal structure of the MthK channel in a Ca²⁺-bound open state was previously determined at 3.3 Å and revealed an octameric gating ring composed of eight intracellular ligand-binding RCK (regulate the conductance of K⁺) domains. It was suggested that Ca²⁺ binding regulates the gating ring conformation, which in turn leads to the opening and closing of the channel. However, at 3.3 AÅ resolution, the molecular details of the structure are not well defined, and many of the conclusions drawn from that structure were hypothetical. Here we have presented high resolution structures of the MthK RCK domain with and without Ca²⁺ bound from three different crystals. These structures revealed a dimeric architecture of the RCK domain and allowed us to visualize the Ca²⁺ binding and protein-protein contacts at atomic detail. The dimerization of RCK domains is also conserved in other RCK-regulated K⁺ channels and transporters, suggesting that the RCK dimer serves as a basic unit in the gating ring assembly. A comparison of these dimer structures confirmed that the dimer interface is indeed flexible as suggested previously. However, the conformational change at the flexible interface is of an extent smaller than the previously hypothesized gating ring movement, and a reconstruction of these dimers into octamers by applying protein-protein contacts at the fixed interface did not generate enclosed gating rings. This indicated that there is a high probability that the previously defined fixed interface may not be fixed during channel gating. In addition to the structural studies, we have also carried out biochemical analyses and have shown that near physiological pH, isolated RCK domains form a stable octamer in solution, supporting the notion that the formation of octameric gating ring is a functionally relevant event in MthK gating. Additionally, our stability studies indicated that Ca²⁺ binding stabilizes the RCK domains in this octameric state.

Received for publication, July 26, 2005 , and in revised form, October 12, 2005.

The atomic coordinates and structure factors (codes 2AEF, 2AEJ, and 2AEM) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the National Institute of Health (Grant GM071621 to Y. J.), Searle Scholars Program (to Y. J.), and the Robert A. Welch Foundation (Grant I-1578 to Y. J.). 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 Both authors contributed equally to this work.

² To whom correspondence should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040. Tel.: 214-645-6027; Fax: 214-645-6042; E-mail: youxing.jiang{at}utsouthwestern.edu.

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