The calycin constellation of the protein universe

Calycins form a large protein superfamily sharing like -barrel structures. Calycins can be divided into several families, among which fatty acid binding proteins and lipocalins. The latter is the largest and functionally the most diverse; lipocalins are extracellular proteins with several common recognition properties such as ligand binding, receptor binding and the formation of complexes with other macromolecules. Through the years our group has studied several aspects of calycins with both in vitro and in silico methods. In this presentation I will review our results about some peculiar aspects of ligand binding by these proteins. During the interaction with their ligands these proteins undergo conformational changes; involvement in this rearrangement of a pKa switch has been reported for some model calycins. In a survey on their electrostatics, we searched for the occurrence in the native structure of calycins of amino acids with anomalous pKa and sought whether a general pattern could be recognized. To this aim, we selected four calycins we had either purified from natural sources or prepared with recombinant DNA technologies. In our survey, we used both in silico prediction methods and in vitro validation procedures, such as isoelectric focusing, electrophoretic titration curves and spectroscopic techniques. By comparing the results under native conditions (no exposure of the proteins to chaotropic agents) to those after protein unfolding (in the presence of 8 M urea), we observed a shift in the pKa of at least one amino acid per protein, which results in a measurable change in pI. Three types of amino acids are involved (Cys, Glu, and His); their position varies along the calycin sequence. No common mechanism may be recognized but we hypothesize that the 'normalization' of anomalous pKa may be the phenomenon that accompanies, and favors, structural rearrangements upon ligand binding also in these calycins. The interaction of calycins with their ligands is often characterized by such an unusual feature as the required presence of water molecules in their calyces. For instance, chicken liver bile acid binding protein (cL-BABP) crystallizes with water molecules in its binding site. To obtain insights on the role of internal water, we performed molecular dynamics (MD) simulations in explicit solvent for cL-BABP, as apo form and as a complex with two molecules of cholic acid, and analyzed in detail the dynamics properties of all water molecules. The diffusion coefficients of the more persistent internal water molecules are significantly different from the bulk, but similar between the two protein forms. A different number of molecules and a different organization are observed for apo- and holo-cL-BABP. Most water molecules identified in the binding site of the apo-crystal diffuse to the bulk during the simulation. In contrast, almost all the internal waters of the holo-crystal maintain the same interactions with internal sidechains and ligands, which suggests they have a relevant role in protein-ligand molecular recognition. Only in the presence of these water molecules we were able to reproduce, by a classical molecular docking approach, the structure of the complex cL-BABP::cholic acid with a low ligand root mean square deviation (RMSD) with respect to its reference positioning. A further feature of calycins is the broad specificity of their binding; we investigated possible in vivo consequences of such behavior in a clinical setup. With the combined use of in vitro (19F-NMR) and in silico (molecular docking) procedures we could demonstrate the affinity of a number of human calycins (lipid-binding proteins from ileum, liver, heart, adipose tissue and epidermis, and retinol-binding protein from intestine) for different drugs (mainly steroids and vastatins). Dissociation constants (Ki) for drugs typically lie in the same range as Ki for natural ligands; in most instances vastatins are the strongest interactors, with atorvastatin ranking top in half of the cases. The affinity of some calycins for some of the vastatins is in the order of magnitude of the drug Cmax after systemic administration in humans. While our data are a proof-of-concept for such interactions, biological implications, if any, are then strongly dependent on a number of complex delivery parameters (route of administration, binding to carrier proteins, distribution to, and accumulation in, human tissues).