12‐Bromododecanoic acid binds inside the calyx of bovine β‐lactoglobulin

Abstract

The X-ray structure of bovine L L-lactoglobulin with the ligand 12-bromododecanoic acid as a model for fatty acids has been determined at a resolution of 2.23 A î in the trigonal lattice Z form. The ligand binds inside the calyx, resolving a long-standing controversy as to where fatty-acid like ligands bind. The carboxylate head group lies at the surface of the molecule, and the lid to the calyx is open at the pH of crystallization (pH 7.3), consistent with the conformation observed in ligand-free bovine L L-lactoglobulin in lattice Z at pH 7.1 and pH 8.2.z 1998 Federation of European Biochemical Societies.

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

“…12, 2008 4273 ratio of 2 according to the previous crystallographic study. 20 We found a weak extra electron density located near the C-terminal R-helical region. Later, we used a rationally designed crystallography experiment for improving electron density of vitamin D 3 of the secondary binding site.…”

“…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.…”

“…Subsequently, we searched for an extra density around the C-terminal R-helix (sites 3 and 4) in the electron density map of the cocrystal prepared in a previous crystallographic study. 20 A weak electron density around site 4 (located between R-helix and -strand I) of LG-vitamin D 3 complex was found. The rationally designed crystallographic approach resulted in obtaining cocrystals with sufficient quality and ligand occupancy so as to allow experimental validation of the second vitamin D 3 binding site on LG.…”

“…18 A remarkable feature of the calyx is its ability to bind hydrophobic molecules such as retinol, fatty acids, and vitamin D 3 ( Figure 1b). [19][20][21][22] The location of vitamin D binding sites has been controversial, yet most evidence points toward the calyx. 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.…”

“…20 A search for an extra electron density for vitamin D 3 around potential secondary binding sites was based on the predicted data obtained from bioinformatic analysis. We found a weak extra electron density that was located near the C-terminal R-helical region.…”