Abstract
GntR/FadR family featuring an N-terminal winged helix-turn-helix DNA-binding domain and a C-terminal α-helical effector-binding and oligomerization domain constitutes one of the largest families of transcriptional regulators. Several GntR/FadR regulators govern the metabolism of sugar acids, carbon sources implicated in bacterial-host interactions. Although effectors are known for a few sugar acid regulators, the unavailability of relevant structures has left their allosteric mechanism unexplored. Here, using DgoR, a transcriptional repressor of d-galactonate metabolism in Escherichia coli, as a model, and its superrepressor alleles, we probed allostery in a GntR/FadR family sugar acid regulator. Genetic and biochemical studies established compromised response to d-galactonate as the reason for the superrepressor behavior of the mutants: T180I does not bind d-galactonate, and while A97V, S171L and M188I bind d-galactonate, effector binding does not induce a conformational change required for derepression, suggesting altered allostery. For mechanistic insights into allosteric communication, we performed simulations of the modeled DgoR structure in different allosteric states for both the wild-type and mutant proteins. We found that each mutant exhibits unique dynamics disrupting the intrinsic allosteric communication pathways, thereby impacting DgoR function. We finally validated the allosteric communication model by testing in silico predictions with experimental data.