Nonmonotonic Modulation of the Protein-Ligand Recognition Event by Inert Crowders

The ubiquitous event of a protein recognizing small molecules or ligands at its native binding site is crucial for initiating major biological processes. However, how a crowded environment, as is typically represented by a cellular interior, would modulate the protein-ligand search process is largely debated. Excluded volume-based theory suggests that the presence of an inert crowder would reinforce a steady stabilization and enhancement of the protein-ligand recognition process. Here, we counter this long-held perspective via the molecular dynamics simulation and Markov state model of the protein-ligand recognition event in the presence of inert crowders. Specifically, we demonstrate that, depending on concentration, even purely inert crowders can exert a nonmonotonic effect via either stabilizing or destabilizing the protein-ligand binding event. Analysis of the kinetic network of binding pathways reveals that the crowders would either modulate precedent non-native on-pathway intermediates or would devise additional ones in a multistate recognition event across a wide range of concentrations. As an important insight, crowders gradually shift the relative transitional preference of these intermediates toward a native-bound state, with ligand residence time at the binding pocket dictating the trend of nonmonotonic concentration dependence by simple inert crowders.