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.