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J. Biol. Chem., Vol. 281, Issue 2, 962-967, January 13, 2006

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Leu³⁰⁹ Plays a Critical Role in the Encapsulation of Substrate Protein into the Internal Cavity of GroEL^*

Ayumi Koike-Takeshita, Tatsuro Shimamura¶, Ken Yokoyama||, Masasuke Yoshida||1, and Hideki Taguchi**

From the Chemical Resources Laboratory, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8503, Japan, the Department of Biological Sciences, Imperial College, London SW7 2AZ, United Kingdom, the ^||ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation, 5800-3 Nagatsuta, Midori-ku, Yokohama 226-0026, Japan, the ^**Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation, 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan, the Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan, and the ^¶Structural Biophysics Laboratory, RIKEN Harima Institute, Spring-8, 1-1-1 Kouto, Mikazuki, Sayo-gun, Hyogo 679-5148, Japan

In the crystal structure of the native GroEL·GroES·substrate protein complex from Thermus thermophilus, one GroEL subunit makes contact with two GroES subunits. One contact is through the H-I helices, and the other is through a novel GXXLE region. The side chain of Leu, in the GXXLE region, forms a hydrophobic cluster with residues of the H helix (Shimamura, T., Koike-Takeshita, A., Yokoyama, K., Masui, R., Murai, N., Yoshida, M., Taguchi, H., and Iwata, S. (2004) Structure (Camb.) 12, 1471-1480). Here, we investigated the functional role of Leu in the GXXLE region, using Escherichia coli GroEL. The results are as follows: (i) cross-linking between introduced cysteines confirmed that the GXXLE region in the E. coli GroEL·GroES complex is also in contact with GroES; (ii) when Leu was replaced by Lys (GroEL(L309K)) or other charged residues, chaperone activity was largely lost; (iii) the GroEL(L309K)·substrate complex failed to bind GroES to produce a stable GroEL(L309K)·GroES·substrate complex, whereas free GroEL(L309K) bound GroES normally; (iv) the GroEL(L309K)·GroES·substrate complex was stabilized with BeF_x, but the substrate protein in the complex was readily digested by protease, indicating that it was not properly encapsulated into the internal cavity of the complex. Thus, conformational communication between the two GroES contact sites, the H helix and the GXXLE region (through Leu³⁰⁹), appears to play a critical role in encapsulation of the substrate.

Received for publication, June 9, 2005 , and in revised form, August 25, 2005.

^* 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 To whom correspondence should be addressed. Fax: 81-45-924-5277; E-mail: myoshida{at}res.titech.ac.jp.

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