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Crystal structure of a secondary vitamin D3 binding site of milk β-lactoglobulin

Authors

  • Ming-Chi Yang,

    1. Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Taiwan, Republic of China
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  • Hong-Hsiang Guan,

    1. Life Science Group, Research Division, National Synchrotron Radiation Research Center, Taiwan, Republic of China
    2. Department of Physics, Institute of Bioinformatics and Structural Biology, National Tsing Hua University,Taiwan, Republic of China
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  • Ming-Yih Liu,

    1. Life Science Group, Research Division, National Synchrotron Radiation Research Center, Taiwan, Republic of China
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  • Yih-Hung Lin,

    1. Life Science Group, Research Division, National Synchrotron Radiation Research Center, Taiwan, Republic of China
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  • Jinn-Moon Yang,

    1. Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Taiwan, Republic of China
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  • Wen-Liang Chen,

    1. Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Taiwan, Republic of China
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  • Chun-Jung Chen,

    Corresponding author
    1. Life Science Group, Research Division, National Synchrotron Radiation Research Center, Taiwan, Republic of China
    2. Department of Physics, Institute of Bioinformatics and Structural Biology, National Tsing Hua University,Taiwan, Republic of China
    • Life Science Group, Research Division, National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu, 30076 Taiwan, ROC
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  • Simon J. T. Mao

    Corresponding author
    1. Department of Biological Science and Technology, College of Biological Science and Technology, National Chiao Tung University, Taiwan, Republic of China
    2. Department of Biotechnology and Bioinformatics, Asian University, Taiwan, Republic of China
    • Department and College of Biological Science and Technology, National Chiao Tung University, 75 Po-Ai Street, Hsinchu, 30068 Taiwan, ROC
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Abstract

β-lactoglobulin (β-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly β structure. The structural function of the only α-helix with three turns at the C-terminus is unknown. Vitamin D3 binds to the central calyx formed by the β-strands. Whether there are two vitamin D binding-sites in each β-LG molecule has been a subject of controversy. Here, we report a second vitamin D3 binding site identified by synchrotron X-ray diffraction (at 2.4 Å resolution). In the central calyx binding mode, the aliphatic tail of vitamin D3 clearly inserts into the binding cavity, where the 3-OH group of vitamin D3 binds externally. The electron density map suggests that the 3-OH group interacts with the carbonyl of Lys-60 forming a hydrogen bond (2.97 Å). The second binding site, however, is near the surface at the C-terminus (residues 136–149) containing part of an α-helix and a β-strand I with 17.91 Å in length, while the span of vitamin D3 is about 12.51 Å. A remarkable feature of the second exosite is that it combines an amphipathic α-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a β-strand providing a nonpolar (Ile-147) and a buried polar residue (Arg-148). They are linked by a hydrophobic loop (Ala-142, Leu-143, Pro-144, and Met-145). Thus, the binding pocket furnishes strong hydrophobic force to stabilize vitamin D3 binding. This finding provides a new insight into the interaction between vitamin D3 and β-LG, in which the exosite may provide another route for the transport of vitamin D3 in vitamin D3 fortified dairy products. Atomic coordinates for the crystal structure of β-LG-vitamin D3 complex described in this work have been deposited in the PDB (access code 2GJ5). Proteins 2008. © 2007 Wiley-Liss, Inc.

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