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

Abstract

Beta-lactoglobulin (beta-LG), one of the most investigated proteins, is a major bovine milk protein with a predominantly beta structure. The structural function of the only alpha-helix with three turns at the C-terminus is unknown. Vitamin D(3) binds to the central calyx formed by the beta-strands. Whether there are two vitamin D binding-sites in each beta-LG molecule has been a subject of controversy. Here, we report a second vitamin D(3) binding site identified by synchrotron X-ray diffraction (at 2.4 A resolution). In the central calyx binding mode, the aliphatic tail of vitamin D(3) clearly inserts into the binding cavity, where the 3-OH group of vitamin D(3) 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 A). The second binding site, however, is near the surface at the C-terminus (residues 136-149) containing part of an alpha-helix and a beta-strand I with 17.91 A in length, while the span of vitamin D(3) is about 12.51 A. A remarkable feature of the second exosite is that it combines an amphipathic alpha-helix providing nonpolar residues (Phe-136, Ala-139, and Leu-140) and a beta-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 D(3) binding. This finding provides a new insight into the interaction between vitamin D(3) and beta-LG, in which the exosite may provide another route for the transport of vitamin D(3) in vitamin D(3) fortified dairy products. Atomic coordinates for the crystal structure of beta-LG-vitamin D(3) complex described in this work have been deposited in the PDB (access code 2GJ5).

β-Lactoglobulin-linoleate complexes: In vitro digestion and the role of protein in fatty acid uptake

“…All members of the lipocalin family contain a β-barrel, shaped into a calyx, composed of eight antiparallel β-strands (Flower, 1996). It has been suggested that βlg binds hydrophobic ligands in its internal calyx, in a crevice near the α-helix on the external surface of the β-barrel and near the interface of βlg dimers (Lange et al, 1998, Wang et al, 1998, Wu et al, 1999, Muresan et al, 2001, Kontopidis et al, 2004, Forrest et al, 2005, Dong et al, 2006, Yang et al, 2008. However, the binding sites and stoichiometries of ligands have been controversial (Wang et al, 1998, Wu et al, 1999, Kontopidis et al, 2002.…”

Rational Design for Crystallization of β-Lactoglobulin and Vitamin D3 Complex: Revealing a Secondary Binding Site

“…Since LG is a molten globule with a heating for 16 min (Table 1). 26 To confirm it further, we monitored the binding of heated LG (100°C for 16 min) with various amounts of vitamin D 3 , and a stoichiometry of 1:1 was observed between heat-denatured LG and vitamin D 3 using the same fluorescence quenching analysis. 26 Taking the previous pH and thermal experiments together (Table 1), we concluded that a thermally stable site (defined as an exosite) beyond the calyx exists for vitamin D 3 binding.…”

“…26 To confirm it further, we monitored the binding of heated LG (100°C for 16 min) with various amounts of vitamin D 3 , and a stoichiometry of 1:1 was observed between heat-denatured LG and vitamin D 3 using the same fluorescence quenching analysis. 26 Taking the previous pH and thermal experiments together (Table 1), we concluded that a thermally stable site (defined as an exosite) beyond the calyx exists for vitamin D 3 binding. In the present study, we located the secondary vitamin D 3 binding site of LG using bioinformatic analysis to narrow the region of potential binding sites, the search of extra electron density around the potential binding sites of the cocrystal prepared on previous crystallographic study, 20 and a rationally designed crystallographic approach for obtaining cocrystals with sufficient quality and ligand occupancy.…”

“…The final refined structure determined using the LG-vitamin D 3 complex prepared at pH 8 with a vitamin D 3 /LG ratio of 3:1 was given in detail and discussed elsewhere. 26 The surface model of the LG-vitamin D 3 complex reveals that vitamin D 3 almost perpendicularly inserts into the calyx cavity, while the other vitamin D 3 is located near the carboxyl terminus of LG (residues 136-149 including part of the R-helix and -strand I) ( Figure 6). It shows the exosite containing most hydrophobic residues fitted parallel with one vitamin D 3 molecule (Figure 6).…”

“…It has been postulated that another site exists, [23][24][25] but it could not be verified by the crystal structure using the vitamin D 2 -LG complex. 10 In a previous study, we first conducted a ligand binding assay using the fluorescence changes by retinol, palmitic LG was 2:1, whereas that of retinol or palmitic acid was 1:1 (Table 1), 26 suggesting that there was another binding site for the vitamin D 3 molecule. Second, we manipulated the binding capability by switching off the gate of calyx at low pH (2)(3)(4)(5)(6) 15 to further substantiate the "two site hypothesis".…”