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Kinetic and Thermodynamic Evidence for Flipping of a Methyl-CpG Binding Domain on Methylated DNA^†

Kosuke Inomata^‡, Izuru Ohki^§, Hidehito Tochio*^‡, Kenichiro Fujiwara, Hidekazu Hiroaki and Masahiro Shirakawa*^‡^#

Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Kyoto 615-8510, Japan, CREST, Japan Science and Technology Corporation, 4-1-8, Honcho, Kawaguchi, Saitama 332-0012, Japan, Division of Structural Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan, Field of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29, Tsurumi, Yokohama, Kanagawa 230-0045, Japan, and Division of Structural Biology, Graduate School of Medicine, Kobe University, 7-5-1, Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan

Biochemistry, 2008, 47 (10), pp 3266–3271

DOI: 10.1021/bi7019029

Publication Date (Web): February 12, 2008

†

This work was supported by grants to M.S. and H.T. from the Japanese Ministry of Education, Science, Sports and Culture and to M.S. from Core Research for Evolution Science and Technology (CREST) of the Japan Science and Technology Agency. This research was also supported in part by the Global COE Program “International Center for Integrated Research and Advanced Education in Materials Science” (B-09) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, administrated by the Japan Society for the Promotion of Science. We acknowledge support by the Global COE Program “Integrated Materials Science” (B-09)

‡

Kyoto University.

Kyushu University.

* To whom correspondence should be addressed. H.T.: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto 615-8510, Japan; phone, +81-75-383-2536 ; fax, +81-75-383-2541; e-mail, tochio@moleng.kyoto-u.ac.jp. M.S.: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Nishikyo-Ku, Kyoto 615-8510, Japan; phone, +81-75-383-2535 ; fax, +81-75-383-2541; e-mail, shirakawa@moleng.kyoto-u.ac.jp.

Yokohama City University.

Kobe University.

Japan Science and Technology Corp.

Abstract

The methyl-CpG binding domain (MBD) is a conserved domain in transcriptional factors that binds to methylated CpG dinucleotide DNA sequences in vertebrates. The complex is comprised of an asymmetric MBD monomer and a symmetric DNA duplex. Therefore, in the complex, each strand of the duplex DNA is in contact with the protein at a distinct surface and thus exhibits a different chemical shift in NMR spectra. Two-dimensional chemical exchange spectroscopy revealed the presence of a stochastic exchange of the two strands of the duplex DNA in the complex at a rate of 4 s⁻¹ at 25 °C, which indicates the existence of a motion of the MBD such that the orientation of the MBD becomes reversed with respect to the DNA duplex. Kinetic and thermodynamic analyses using surface plasmon resonance, quartz crystal microbalance, and isothermal titration calorimetry suggest that the reversal of MBD with respect to the DNA duplex takes place without its complete dissociation from DNA, indicating the presence of an intermediate protein−DNA binding state that allows the protein to undergo a flip motion upon DNA.

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