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Originally published In Press as doi:10.1074/jbc.M213164200 on February 7, 2003

J. Biol. Chem., Vol. 278, Issue 16, 14211-14218, April 18, 2003
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Three-dimensional Localization of Divergent Region 3 of the Ryanodine Receptor to the Clamp-shaped Structures Adjacent to the FKBP Binding Sites*

Jing ZhangDagger , Zheng Liu§, Haruko MasumiyaDagger , Ruiwu WangDagger , Dawei JiangDagger ||, Fei Li§, Terence Wagenknecht§**, and S. R. Wayne ChenDagger Dagger Dagger

From the Dagger  Cardiovascular Research Group, Departments of Physiology and Biophysics and of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada, the § Wadsworth Center, New York State Department of Health, Albany, New York 12201, and the ** Department of Biomedical Sciences, School of Public Health, State University of New York, Albany, New York 12201

Of the three divergent regions of ryanodine receptors (RyRs), divergent region 3 (DR3) is the best studied and is believed to be involved in excitation-contraction coupling as well as in channel regulation by Ca2+ and Mg2+. To gain insight into the structural basis of DR3 function, we have determined the location of DR3 in the three-dimensional structure of RyR2. We inserted green fluorescent protein (GFP) into the middle of the DR3 region after Thr-1874 in the sequence. HEK293 cells expressing this GFP-RyR2 fusion protein, RyR2T1874-GFP, were readily detected by their green fluorescence, indicating proper folding of the inserted GFP. RyR2T1874-GFP was further characterized functionally by assays of Ca2+ release and [3H]ryanodine binding. These analyses revealed that RyR2T1874-GFP functions as a caffeine- and ryanodine-sensitive Ca2+ release channel and displays Ca2+ dependence and [3H]ryanodine binding properties similar to those of the wild type RyR2. RyR2T1874-GFP was purified from cell lysates in a single step by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The three-dimensional structure of the purified RyR2T1874-GFP was then reconstructed using cryoelectron microscopy and single particle image analysis. Comparison of the three-dimensional reconstructions of wild type RyR2 and RyR2T1874-GFP revealed the location of the inserted GFP, and hence the DR3 region, in one of the characteristic domains of RyR, domain 9, in the clamp-shaped structure adjacent to the FKBP12 and FKBP12.6 binding sites. COOH-terminal truncation analysis demonstrated that a region between 1815 and 1855 near DR3 is essential for GST-FKBP12.6 binding. These results provide a structural basis for the role of the DR3 region in excitation-contraction coupling and in channel regulation.

* This work was supported by research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, N.W.T. and Nunavut (to S. R. W. C.) and by the Muscular Dystrophy Association and National Institutes of Health Grant AR40615 (to T. W.). The Resource for Visualization of Biological Complexity was supported by National Institutes of Health Biotechnological Resource Grant RR01219.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported by the Uehara Memorial Foundation (Japan) and Postdoctoral Fellowships from the Heart and Stroke Foundation of Canada and the Alberta Heritage Foundation for Medical Research (AHFMR).

|| Recipient of the Alex W. Church Graduate Student Award.

Dagger Dagger AHFMR Senior Scholar. To whom correspondence should be addressed. Tel.: 403-220-4235; Fax: 403-283-4841; E-mail: swchen@ ucalgary.ca.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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