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

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 D ₃ 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).