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

Abstract

-Lactoglobulin (LG) is a major milk whey protein containing primarily a calyx for vitamin D 3 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 D 3 in the complex by altering the pH and initial ratios of vitamin D 3 /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 D 3 /LG (3:1) are crucial to optimize the occupancy and enhance the electron density of vitamin D 3 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.

Parallel Screening of Wild-Type and Drug-Resistant Targets for Anti-Resistance Neuraminidase Inhibitors

“…Our previous studies revealed that the performance of GEMDOCK is similar to other docking methods such as DOCK [26], FlexX [27], and GOLD [25], [28], [29]. Furthermore, GEMDOCK has been successfully applied to identify novel inhibitors and binding sites for several targets [30]–[32]. After the docking procedure, we used the docked compounds to characterize the mutant subsite using site-moiety maps [23], which present the relationship between moiety preferences and physico-chemical properties of the binding site through anchors (Fig.…”

Pathway-based Screening Strategy for Multitarget Inhibitors of Diverse Proteins in Metabolic Pathways

“…Our previous studies revealed that GEMDOCK has similar performance to other docking methods such as DOCK [21], FlexX [22], and GOLD [20], [23], [24]. Furthermore, we have successfully used GEMDOCK to identify novel inhibitors and binding sites for several targets [25]–[27]. Subsequently, the site-moiety maps of SDH and SK were established by statistical analysis of the top 6,000 docked compounds (approximately 2% of the 302,909 compounds) (Fig.…”

Addition of β-Lactoglobulin Produces Water-Soluble Shikonin

“…29−31 Some studies suggest that there might also be an alternative binding site located on the outer surface of the protein. 30,32 β-Lg can also be used as an entrapment agent for some compounds of interest, such as (−)-epigallocatechin-3-gallate; an entrapped secondary metabolite shows higher stability in the form of produced nanoparticles, and consequently its activity is preserved. 33 The binding of a ligand to the calyx is not only a function of protein structure and ligand size but also depends on extrinsic parameters such as the medium pH.…”