Substrate selectivity is an important preventive measure to decrease the possibility of cross interactions between enzymes and metabolites that share structural similarities. In addition, understanding the mechanisms that determine selectivity towards a particular substrate increases the knowledge base for designing specific inhibitors for target enzymes. Here, we combine NMR, molecular dynamics (MD) simulations, and protein engineering to investigate how two substrate analogues, allylicphosphonate (cPEP) and sulfoenolpyruvate (SEP), recognize the mesophilic (eEIC) and thermophilic (tEIC) homologues of the receptor domain of bacterial Enzyme I, which has been proposed as a target for antimicrobial research. Chemical Shift Perturbation (CSP) experiments show that cPEP and SEP recognize tEIC over the mesophilic homologue. Combined Principal Component Analysis of half-microsecond-long MD simulations reveals that incomplete quenching of a breathing motion in the eEIC-ligand complex destabilizes the interaction and makes the investigated substrate analogues selective toward the thermophilic enzyme. Our results indicate that residual protein motions need to be considered carefully when optimizing small molecule inhibitors of EI. In general, our work demonstrates that protein conformational dynamics can be exploited in the rational design and optimization of inhibitors with subfamily selectivity.
Keywords: MD simulations; NMR; drug discovery; ligand binding; phosphoenolpyruvate; principal component analysis; selective inhibition.