Rational design for crystallization of β-lactoglobulin and vitamin D₃ complex: Revealing a secondary binding site

β-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D₃ binding, although the existence of another site beyond the calyx is controversial. Using fluorescence spectral analyses in the previous study, we showed the binding stoichiometry for vitamin D ₃ to LG to be 2:1 and a stoichiometry of 1:1 when the calyx was "disrupted" by manipulating the pH and temperature, suggesting that a secondary vitamin D binding site existed. To help localize this secondary site using X-ray crystallography in the present study, we used bioinformatic programs (Insight II, Q-SiteFinder, and GEMDOCK) to identify the potential location of this site. We then optimized the occupancy and enhanced the electron density of vitamin D₃ in the complex by altering the pH and initial ratios of vitamin D₃/LG in the cocrystal preparation. We conclude that GEMDOCK can aid in searching for an extra density map around potential vitamin D binding sites. Both pH (8) and initial ratio of vitamin D₃/LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D₃ in the complex for rational-designed crystallization. The strategy in practice may be useful for future identification of a ligand-binding site in a given protein.