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J. Biol. Chem., Vol. 283, Issue 19, 13165-13173, May 9, 2008

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Structural and Thermodynamic Analyses of Solute-binding Protein from Bifidobacterium longum Specific for Core 1 Disaccharide and Lacto-N-biose I^*

Ryuichiro Suzuki¹, Jun Wada^¶12, Takane Katayama^¶, Shinya Fushinobu³, Takayoshi Wakagi, Hirofumi Shoun, Hayuki Sugimoto^||, Akiyoshi Tanaka^||, Hidehiko Kumagai^¶, Hisashi Ashida, Motomitsu Kitaoka^**, and Kenji Yamamoto

From the Department of Biotechnology, the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, ^¶Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa 921-8836, ^||Graduate School of Bioresources, Mie University, Tsu, Mie 514-8507, and ^**National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan

Recently, a gene cluster involving a phosphorylase specific for lacto-N-biose I (LNB; Galβ1–3GlcNAc) and galacto-N-biose (GNB; Galβ1–3GalNAc) has been found in Bifidobacterium longum. We showed that the solute-binding protein of a putative ATP-binding cassette-type transporter encoded in the cluster crystallizes only in the presence of LNB or GNB, and therefore we named it GNB/LNB-binding protein (GL-BP). Isothermal titration calorimetry measurements revealed that GL-BP specifically binds LNB and GNB with K_d values of 0.087 and 0.010 µM, respectively, and the binding process is enthalpy-driven. The crystal structures of GL-BP complexed with LNB, GNB, and lacto-N-tetraose (Galβ1–3GlcNAcβ1–3Galβ1–4Glc) were determined. The interactions between GL-BP and the disaccharide ligands mainly occurred through water-mediated hydrogen bonds. In comparison with the LNB complex, one additional hydrogen bond was found in the GNB complex. These structural characteristics of ligand binding are in agreement with the thermodynamic properties. The overall structure of GL-BP was similar to that of maltose-binding protein; however, the mode of ligand binding and the thermodynamic properties of these proteins were significantly different.

Received for publication, November 29, 2007 , and in revised form, March 7, 2008.

The atomic coordinates and structure factors (code 2Z8D, 2Z8E, and 2Z8F) 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 in part by the Program for Promotion of Basic Research Activities for Innovative Biosciences. 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 a supplemental Table.

¹ Both authors contributed equally to this work.

² Supported by a grant from the 21st Century COE Program of the Ministry of Education, Culture, Sports, Science, and Technology, Japan, to the Graduate School of Biostudies and Institute for Virus Research, Kyoto University.

³ To whom correspondence should be addressed. Fax: 81-3-5841-5151; E-mail: asfushi{at}mail.ecc.u-tokyo.ac.jp.

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