Computational studies on bovine beta-lactoglobulin

In the last years our research group has focused its attention on bovine beta-lactoglobulin as a model protein of the lipocalin superfamily, extracellular proteins with the ability to bind small hydrophobic molecules. The understanding of its structural and binding features at a molecular level was mandatory in order to clarify the roles of this protein in controlling physiological processes. Bovine beta-lactoglobulin adopts a beta-barrel fold, characterized by a central calyx with an internal hydrophobic surface. Its secondary structure is built up by 8 subsequent antiparallel beta strands, an alpha helix, and a short C-terminal beta-strand. Some hypotheses have been proposed for the biological role of bovine beta-lactoglobulin (fatty acid carrier, activator of the pregastric lipase), but its real function still remains to be clarified. It is known to bind fatty acids, but also cholesterol, retinol, and sex hormones, such as estrogens and progestogens. Different computational studies supported by experimental results were carried out on this protein by our group, and the collected evidence was compared with data on other proteins of the same superfamily. It is well known that bovine beta-lactoglobulin presents a pH-dependent conformational change. We run some classical molecular dynamics simulations, combined with a sampling of protonation states of all its titrable sites based on continuum electrostatics/Monte Carlo. In this specific application, we performed the continuum electrostatics calculation solving the Poisson–Boltzmann equation by a finite differences two-step focusing procedure. The continuum electrostatics energy terms thus obtained were used for the Monte Carlo sampling of protonation states, eventually providing us the pKa and pKhalf for each residue during the simulations. We also investigated the ability of bovine beta-lactoglobulin to bind drugs of different classes, and compared the efficiency of rigid vs induced-fit docking approach based on a Monte Carlo sampling and simulated annealing. The binding free energies of the complexes were then computed by an empirical scoring function, and the results were validated by comparing them with experimental data obtained through NMR experiments. No water molecules are present in the binding site (calyx) of bovine beta-lactoglobulin either in its apo- or in its holo-form. So we analyzed if this peculiar behavior is common to other proteins belonging to the lipocalin superfamily, by analyzing in detail the chicken liver bile acid binding protein. We wrote a program based on the Delaunay tessellations, in order to identify all the water molecules present in the protein binding site during the molecular dynamics simulations. We identified a set of 'constitutive' water molecules and eventually, through a classical molecular docking approach, we demonstrated that these ones are mandatory in the protein::ligand molecular recognition mechanism. More recently we are extending the electrostatics evaluations carried out on bovine beta-lactoglobulin to other proteins of the same superfamily in order to understand if the anomalous pKhalf of Glu89 described for beta-lactoglobulin is just an isolated case or if it featurs a shared mechanism of the proteins of this superfamily. To achieve our purpose, we combined experimental electrophoretic techniques to LowMode molecular dynamics simulations and an empirical method for the calculation of pKa shifts based on the PROPKA approach. Data confirmed that peculiarities in the electrostatics of the native states of some calycins, as we have described, do not reflect an identical behavior of some specific aminoacids: both the chemical nature and the position of the aminoacids with anomalous pKa vary from a protein to another, and the width of the pKa shifts and their compound effect on pI also vary quite extensively.