Macromolecular Crowder and Ligand Compete for the Closed Domain Cleft of Maltose Binding Protein

Abstract

Protein-ligand binding is central to many biochemical processes including enzyme catalysis/inhibition and cellular signaling. In the cellular context, these processes occur in milieus crowded with bystander macromolecules. Growing evidence suggests that the macromolecular crowders are not inert but influence the biochemical processes. A previous study of our lab demonstrated that a synthetic polymer crowder, Ficoll70, and a ligand, maltose, compete for binding with the maltose binding protein (MBP), a periplasmic protein involved in nutrient uptake and chemotaxis [Miklos and Zhou, PLoS ONE 8, e74969 (2013)]. Fluorescence and NMR spectroscopy showed that Ficoll70 weakly binds to MBP and this binding is abrogated by saturating amounts of maltose. MBP has a bi-lobed structure; the lobes close up upon ligand binding into the domain cleft. Competitive inhibition by Ficoll70 could result from the crowder preferentially binding either to the open cleft, thus rendering the protein incompetent for maltose binding, or to the closed cleft, thus blocking maltose from the binding site. To distinguish between these two possibilities, here we introduced MBP hinge mutations that result in cleft closure and enhancement of maltose binding affinity. With the closure mutants, MBP-Ficoll70 interactions would be expected to weaken in the open-cleft binding scenario but to be unperturbed or even strengthened in the closed-cleft binding scenario. Our data indicate a modest increase in Ficoll70 binding affinity for a closure mutant, thus supporting the closed-cleft binding scenario. A panel of mutants with varying degrees of cleft closure is being studied to validate the observation. Our study will provide new insight into the nonrandom nature of weak protein-crowder interactions in cellular environments.