Influence of physicochemical factors on environmental availability and distribution of semiochemicals that affect Varroa destructor and phylogenetically close organisms: classification by VHWOC PCA-clustering.

Varroa destructor parasites Apis mellifera larvae following the interception of the semiochemicals involved in bee communication; thus, the semiochemical availability and distribution pathways take place within different physicochemical environments. The structure of 172 molecules with semiochemical activity on Varroa destructor was used to compute the representative physicochemical descriptors of the thermodynamic partition among different physicochemical environments: vapor pressure (V), Henry's coefficient (H), water solubility constant (W), octanol-water partition coefficient (O) and organic carbon partition coefficient (C); VHWOC. The principal component analysis (PCA) and hierarchical clustering of VHWOC descriptors allowed us to establish the trend in availability and distribution of the semiochemicals resulting in a 4 classes model of physicochemical environments: Class 1, Soluble/Volatile; Class 2, Soluble; Class 3, Contact; Class 4, Adsorbed/Volatile. Our results suggest that semiochemicals can transit between different thermodynamic equilibrium phases depending on environment conditions. The classification prediction of the model was tested on 6 new molecules obtained from ketonic extracts of L5 Apis mellifera drone larvae; locating them in class 4, which was consistent with their molecular structure. This study can be the starting point for the design of synthetic semiochemicals or for the control of Varroa destructor. In addition, the method can be used in the analysis of other semiochemical groups.

ß-lactoglobulin's conformational requirements for ligand binding at the calyx and the dimer interphase: a flexible docking study.

ß-lactoglobulin (BLG) is an abundant milk protein relevant for industry and biotechnology, due significantly to its ability to bind a wide range of polar and apolar ligands. While hydrophobic ligand sites are known, sites for hydrophilic ligands such as the prevalent milk sugar, lactose, remain undetermined. Through the use of molecular docking we first, analyzed the known fatty acid binding sites in order to dissect their atomistic determinants and second, predicted the interaction sites for lactose with monomeric and dimeric BLG. We validated our approach against BLG structures co-crystallized with ligands and report a computational setup with a reduced number of flexible residues that is able to reproduce experimental results with high precision. Blind dockings with and without flexible side chains on BLG showed that: i) 13 experimentally-determined ligands fit the calyx requiring minimal movement of up to 7 residues out of the 23 that constitute this binding site. ii) Lactose does not bind the calyx despite conformational flexibility, but binds the dimer interface and an alternate Site C. iii) Results point to a probable lactolation site in the BLG dimer interface, at K141, consistent with previous biochemical findings. In contrast, no accessible lysines are found near Site C. iv) lactose forms hydrogen bonds with residues from both monomers stabilizing the dimer through a claw-like structure. Overall, these results improve our understanding of BLG's binding sites, importantly narrowing down the calyx residues that control ligand binding. Moreover, our results emphasize the importance of the dimer interface as an insufficiently explored, biologically relevant binding site of particular importance for hydrophilic ligands. Furthermore our analyses suggest that BLG is a robust scaffold for multiple ligand-binding, suitable for protein design, and advance our molecular understanding of its ligand sites to a point that allows manipulation to control binding.

Role of lysine epsilon-amino groups of beta-lactoglobulin on its activating effect of Kluyveromyces lactis beta-galactosidase.

Native beta-lactoglobulin binds and increases the activity of Kluyveromyces lactis beta-galactosidase. Construction of a three-dimensional (3D) model of beta-lactoglobulin showed that lysine residues 15, 47, 69, and 138 are the most exposed ones, thus the ones more likely to interact with beta-galactosidase. Molecular docking estimated the interaction energies of amino acid residues with either lactose or succinic anhydride, showing that Lys(138) is the most likely to react with both. Affinity chromatography demonstrated that succinylated beta-lactoglobulin diminished its ability to bind to the enzyme. Furthermore, when activity was measured in the presence of succinylated beta-lactoglobulin, its activating effect was lost. Since succinylation specifically blocks Lys epsilon-amino groups, their loss very likely causes the disappearance of the activating effect. Results show that the activating effect of beta-lactoglobulin on beta-galactosidase activity is due to the interaction between both proteins and that this interaction is very likely to occur through the Lys epsilon-amino groups of beta-lactoglobulin.