Promiscuous Binding of Ligands by β-Lactoglobulin Involves Hydrophobic Interactions and Plasticity

Bovine β-lactoglobulin (βLG) binds a variety of hydrophobic ligands, though precisely how is not clear. To understand the structural basis of this promiscuous binding, we studied the interaction of βLG with palmitic acid (PA) using heteronuclear NMR spectroscopy. The titration was monitored using tryptophan fluorescence and a HSQC spectrum confirmed a 1:1 stoichiometry for the PA-βLG complex. Upon the binding of PA, signal disappearances and large changes in chemical shifts were observed for the residues located at the entrance and bottom of the cavity, respectively. This observation indicates that the lower region makes a rigid connection with PA whereas the entrance is more flexible. The result is in contrast to the binding of PA to intestinal fatty acid-binding protein, another member of the calycin superfamily, in which structural consolidation occurs upon ligand binding. On the other hand, the ability of βLG to accommodate various hydrophobic ligands resembles that of GroEL, in which a large hydrophobic cavity and flexible binding site confer the ability to bind various hydrophobic substrates. Considering these observations, it is suggested that, in addition to the presence of the hydrophobic cavity, the plasticity of the entrance region makes possible the binding of hydrophobic ligands of various shapes. Thus, in contrast to the specific binding seen for many enzymes, βLG provides an example of binding with low specificity but high affinity, which may play an important role in protein–ligand and protein–protein networks.