Allosteric regulatory control in dihydrofolate reductase (DHFR) revealed with dynamic asymmetry in unliganded ensemble.

Abstract

We investigate how distal mutations modulate the dynamics of functional sites of Escherichia coli dihydrofolate reductase (DHFR) leading to an impact on activity using our computational dynamics toolset. Conformations of functionally important loops of DHFR, namely the M20 & FG loops, were previously shown to be impacted by mutations at distal positions. We examined the dynamics of these loops in wild type unliganded DHFR by combining long molecular dynamics simulation with position-specific Dynamic Flexibility Index (DFI) and Dynamic Coupling Index (DCI) metrics. The dynamic metrics were compared with the preexisting deep mutational scanning experiment. The results showed DFI metric can identify positions that would be detrimental/beneficial to function upon mutations. The DCI metric, which determines the strength of dynamic coupling of positions with M20 & FG loops, showed these loops exhibit different patterns in mediating long-distance communication with the rest of the protein. This difference implies that substitutions on distal positions coupled to M20 & FG loops can have a distinct impact on each loop; thus, targeting both loops, mutations could possibly be used to generate a diverse complement of functional DHFR variants. To further understand this behavior, the asymmetry in dynamic coupling with M20 & FG loops was examined using the DCIasym metric. This analysis suggested distal residues that dominated the coupling with both loops (which we call “controller” residues) are less prone to substitutions, naturally highly evolving, and substitutions on these residues generally enhances activity. Conversely, substitutions on residues dominated by the coupling of both loops (“controlled” residues) display deleterious functional outcomes. Overall, our results suggest that dynamics-based metrics, particularly asymmetry in dynamic coupling, allow us to identify distal residue positions that could be targeted to rationally engineer enzymes with enhanced activities.