β-Lactoglobulin's Conformational Requirements for Ligand Binding at the Calyx and the Dimer Interphase: a Flexible Docking Study

Abstract

β-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.

Dicarboxyl-terminated iron(ii) clathrochelates as ICD-reporters for globular proteins

“…BLG with its molecular mass of 18.3 kDa contains 162 amino acid residues and its main binding site is postulated to be at its calyx entrance. An accessibility of the calyx for a guest molecule is known 44 to be a pH dependent: at pH lower than 6.5, the EF loop, a motif of this macromolecule containing the residues from I 84 to N 90, remains closed and it opens up at pH >7. 44 The results of the performed molecular docking suggest that each of the clathrochelate molecules is able to t the entrance of the BLG cavity, in which it forms the bonds with amino acid residues of the protein (mainly through the nonpolar dispersion interactions).…”

“…An accessibility of the calyx for a guest molecule is known 44 to be a pH dependent: at pH lower than 6.5, the EF loop, a motif of this macromolecule containing the residues from I 84 to N 90, remains closed and it opens up at pH >7. 44 The results of the performed molecular docking suggest that each of the clathrochelate molecules is able to t the entrance of the BLG cavity, in which it forms the bonds with amino acid residues of the protein (mainly through the nonpolar dispersion interactions). To check an ability of the polar interactions between the amino acid residues and the clathrochelate molecule, we also performed its docking procedure with the residues Leu 46, Leu 54, Ile 56, Ile 71, Ile 84, Phe 105, Met 107, Lys 60, and Lys 69 set as exible.…”

Docking and Molecular Dynamics Predictions of Pesticide Binding to the Calyx of Bovine β-Lactoglobulin

“…It belongs to the lipocalin family of small extracellular proteins that carry hydrophobic ligands [11]. BLG is relevant to the food industry due to its binding spectrum and abundance in cow's milk; thus, it has been extensively characterized, biochemically and structurally, through X-ray diffraction (XRD), NMR and computational approaches [12][13][14][15].…”

“…Each BLG monomer consists of 162 residues (18.3 kDa), folded into eight-stranded antiparallel β-sheets that form a hydrophobic binding pocket called the calyx (shown in Figure 1 and Figure S1), flanked by an α-helix [16]. BLG binds to a variety of nutrients like fatty acids, peptides, sugars and some vitamins ( Figure S2) in at least two different binding sites, the most prominent of which is the calyx [15,17,18] (the second site is formed in the interphase between two BLGs when they dimerize [15], and will not be explored here). The affinity for the known hydrophobic ligands in the calyx is in the micromolar range [19].…”

Revealing the Dimeric Crystal and Solution Structure of β-Lactoglobulin at pH 4 and Its pH and Salt Dependent Monomer–Dimer Equilibrium

“…9,10,14,15 Crystal and NMR structures revealed that BLG has a hydrophobic core of eight antiparallel β-strands (A-H) forming a β-barrel (or calyx) showing a pH dependent accessibility mediated via a conformational change of the flexible loop connecting strands E and F (EF-loop-residues 85-90). 10,16 At pH >7.0 this loop adopts an open conformation as seen in different crystal structures (1BEB, 1BSY, 1B0O, 2Q2M, 2BLG), 1,14,1719 whereas at pH <7.0 a closed form is found in crystal and NMR structures (1CJ5, 2AKQ and 3BLG). 19,20,21 In solution BLG is present in different oligomerization states depending on pH, ionic strength and temperature.…”