Macromolecular crowding effects on coupled folding and binding.

Replica exchange molecular dynamics simulations are performed on the protein complex pKID-KIX to understand the effects of macromolecular crowding on coupled folding and binding events. A structure-based protein model at the residue level is adopted for the two proteins to include intramolecular conformational flexibility, while crowding macromolecules are represented as spherical particles. The interactions between crowders and protein residues can be either purely repulsive or a combination of short-range repulsion and intermediate-range attraction. Consistent with previous studies on rigid-body protein binding in the presence of spherical crowders, we find that the complex formation is stabilized by repulsive protein-crowder interactions and destabilized by sufficiently strong attractive protein-crowder interactions. Competition between stabilizing repulsive and destabilizing attractive interactions is quantitatively captured by a previous theoretical model developed for describing the change in the binding free energy of rigid proteins in a crowded environment. We find that protein flexibility has little effect on the thermodynamics of the pKID-KIX binding (with respect to bulk) for repulsive and weakly attractive protein-crowder interactions. For strong protein-crowder attractive interactions, the destabilizing effect due to crowding is attenuated by protein flexibility. Interestingly, the mechanism of coupled folding and binding observed in bulk remains unchanged under highly crowded conditions over a broad range of protein-crowder interaction strengths. Also, strong protein-crowder attractive interactions can significantly stabilize intermediate states involving partial contact between pKID and KIX domains.