Both Ligands and Macromolecular Crowders Preferentially Bind to Closed Conformations of Maltose Binding Protein.

In cellular environments, proteins not only interact with their specific partners but also encounter a high concentration of bystander macromolecules, or crowders. Nonspecific interactions with macromolecular crowders modulate the activities of proteins, but our knowledge about the rules of nonspecific interactions is still very limited. In previous work, we presented experimental evidence that macromolecular crowders acted competitively in inhibiting the binding of maltose binding protein (MBP) with its ligand maltose. Competition between a ligand and an inhibitor may result from binding to either the same site or different conformations of the protein. Maltose binds to the cleft between two lobes of MBP, and in a series of mutants, the affinities increased with an increase in the extent of lobe closure. Here we investigated whether macromolecular crowders also have a conformational or site preference when binding to MBP. The affinities of a polymer crowder, Ficoll70, measured by monitoring tryptophan fluorescence were 3-6-fold higher for closure mutants than for wild-type MBP. Competition between the ligand and crowder, as indicated by fitting of titration data and directly by nuclear magnetic resonance spectroscopy, and their similar preferences for closed MBP conformations further suggest the scenario in which the crowder, like maltose, preferentially binds to the interlobe cleft of MBP. Similar observations were made for bovine serum albumin as a protein crowder. Conformational and site preferences in MBP-crowder binding allude to the paradigm that nonspecific interactions can possess hallmarks of molecular recognition, which may be essential for intracellular organizations including colocalization of proteins and liquid-liquid phase separation.