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Crystal structure of β‐lactoglobulin and vitamin D complex. Identification of a second binding site for vitamin D.
Author(s) -
Yang Ming Chi,
Chen Chun Jung,
Liu Ming Yih,
Mao Simon JT
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb59-c
Subject(s) - chemistry , vitamin , ramachandran plot , binding site , palmitic acid , retinol binding protein , crystallography , stereochemistry , crystal structure , molecule , protein structure , biochemistry , retinol , fatty acid , organic chemistry
β‐lactoglobulin (β‐LG) is a bovine milk protein sensitive to thermal denaturation. The unique feature of β‐LG is its ability to bind and transport hydrophobic ligands such as retinol, palmitic acid, and vitamin D. Using the fluorescence quenching, we and the others show the maximal binding ratio of β‐LG with vitamin D to be 1:2. It suggests that there are two vitamin D binding sites in each β‐LG molecule, but the issue still remains controversial. In this study, we further show that the second binding site is thermally stable that is only specific for vitamin D, but not for retinol and palmitic acid. Using synchrotron X‐ray diffraction on the crystal of β‐LG‐vitamin D complex at a high resolution 2.2 ?(space group P3221 with unit‐cell parameters a = 53.7 ? b = 53.7 ? c = 111.6 ? alpha = 90? beta = 90? and gamma = 120?, we show that the structure of β‐LG‐vitamin D3 complex with a R value of 0.2473 (Rfree = 0.2978). While, one vitamin D3 molecule binds to the calyx (central binding site) of β‐LG and the other binds to a specific exosite (second binding site). We demonstrate that the second binding site is located near the surface of C‐terminal α‐helix and β‐strand I, where the OH group of vitamin D3 interacts with η1 N of Arg‐148 and δ1 O of Asp‐137, whereas the carbon backbone of vitamin D3 interacts with Phe‐136, Asp‐137, Leu‐140, Lys‐141, Met‐145 and Arg‐148. The significance and physiologic role of this additional binding site in vitamin D supplemented milk will be discussed.