*3.3. Molecular Modeling*

Details on how protein glycation with X impacts further flavonoids binding were collected by means of an in silico approach. The potential binding sites for X were predicted for both α-LA and β-LG molecules by performing molecular docking simulations. The best three fits involving α-LA and β-LG as receptors, decided based on the binding energy values and the interface area, were further analyzed in detail (Table 1). In the case of α-LA, mainly two binding sites located on the protein surface appeared to accommodate the ligands used in the study. The highest affinity of the receptor for its ligand was estimated based on the lowest binding energy. Thus, α-LA exhibited the highest affinity toward X molecules when bound to the cavity involving residues of the α-LA core (Phe53, Gln54, Tyr103, Trp104) and the amino-terminal section of the Leu105-Leu110 helix. In addition to the hydrophobic contacts, three hydrogen bonds of 3.68 Å, 2.37 Å, and 2.83 Å involving Thr33, Leu105 and Ala106, respectively, contributed to the attraction between α-LA and the X molecule hosted within this cavity. Two different relative binding positions of the X molecules with respect to the α-LA receptor, sharing common amino acid residues in contact with the ligand, were predicted with high scores. None of the two X binding modes appeared to affect the attachment of the main flavonoids prevailing in the onion skin extract, namely quercetin-4 -*O*-monoglucoside (QMG) and quercetin-3,4 -*O*-diglucoside (QDG) [41], to the α-LA. In agreement with Horincar et al. [41], the α-LA molecule accommodates, with high specificity, the same binding site of both QDG and QMG ligands (Figure 1). The amino acids establishing direct contacts with QMG are Leu3, Glu11, Leu12, Lys13, Asp14, Thr38, Leu52, Leu85, Thr86, Asp88, Ile89, Met90 and Lys93, whereas the residues responsible for the QDG binding are Glu1, Leu3, Arg10, Glu11, Leu12, Lys13, Thr38, Leu52, Asp83, Leu85, Thr86, Asp88 and Ile<sup>89</sup> [41].

In addition, this wide pocket with a volume of 435.8 Å3 appears to be able to accommodate an X molecule, which overlaps the QMG without interfering with QDG binding. It should be noted that α-LA shows a better affinity towards QMG and QDG (binding energy of −24.21 kcal/mol and −32.01 kcal/mol, respectively) with respect to X (binding energy of −7.48 kcal/mol).

On the other hand, the β-LG molecule is able to accommodate the X molecule in three different pockets with volumes ranging from 137.86 to 217.15 Å (Table 1), without interfering with QMG or QDG binding (Figure 1).

**Table 1.** Molecular details on the interactions between the main whey proteins (α-lactalbumin (α-LA) and β-lactoglobulin monomer (β-LG)) equilibrated at 25 ◦C, xylose (X) and the major flavonoids from onion skins (quercetin-4 -*O*-monoglucoside (QMG) and quercetin-3,4 -*O*-diglucoside (QDG) [41].


β-LG binds the X molecules more tightly compared to α-LA; the X binding energy by the β-LG monomer varies between −16.41 and −13.00 kcal/mol. In addition, the binding of two X molecules to the β-LG pockets involves hydrogen bonds established with Glu<sup>158</sup> in the case of complex 1 and Met24, Asp137 and Leu149 in the case of complex 2, as presented in Table 1. The in silico results successfully complement the experimental findings, adding valuable details on how whey protein glycation with X further impacts flavonoid binding. These atomic level observations indicate that, upon glycation, the β-LG molecule might play a major role in flavonoid biding.

**Figure 1.** Superposition of the models showing the most probable complexes formed between (**a**) α-lactalbumin and (**b**) β-lactoglobulin (represented in blue in surf style) and xylose (represented in orange in licorice style—models 1 and 2), quercetin-4 -*O*-monoglucoside (represented in red in licorice style) and quercetin-3,4 -*O*-diglucoside (represented in green in licorice style). Images were prepared using VMD software [42].
