Structural and Functional Analyses of the Transcription Repressor DgoR From Escherichia coli Reveal a Divalent Metal-Containing D-Galactonate Binding Pocket

Abstract

The transcription repressor of D-galactonate metabolism, DgoR, from Escherichia coli belongs to the FadR family of the GntR superfamily. In the presence of D-galactonate, DgoR binds to two inverted repeats overlapping the dgo cis-acting promoter repressing the expression of genes involved in D-galactonate metabolism. To further understand the structural and molecular details of ligand and effector interactions between Dgalactonate and this FadR family member, herein we solved the crystal structure of C-terminal domain of DgoR (DgoR_C), which revealed a unique divalent metalcontaining substrate binding pocket. The metal ion is required for D-galactonate binding, as evidenced by the dramatically decreased affinity between D-galactonate and DgoR in the presence of EDTA, which can be reverted by the addition of Zn 2+ , Mg 2+ , and Ca 2+. The key amino acid residues involved in the interactions between Dgalactonate and DgoR were revealed by molecular docking studies and further validated with biochemical studies by site-directed mutagenesis. It was found that changes to alanine in residues R102, W181, T191, and R224 resulted in significantly decreased binding affinities for D-galactonate, as determined by EMSA and MST assays. These results suggest that the molecular modifications induced by a D-galactonate and a metal binding in the DgoR are required for DNA binding activity and consequently, transcriptional inhibition.

Identification of a Potential Antimycobacterial Drug Sensitizer Targeting a Flavin-Independent Methylenetetrahydrofolate Reductase

“…To measure the binding affinities between wild-type Rv2172c, wild-type MSMEG_6649, and its site-directed mutants with NADH or compound AB131 or compound 13 or 14 , the MST binding assays were carried out as described previously . Briefly, the NT- 647-NHS dye from the Monolith NTTM Protein Labeling Kit RED-NHS was first used to label the wild-type Rv2172c, wild-type MSMEG_6649, and their mutants by following the manufacturer’s instructions (NanoTemper Technologies).…”

“…To measure the binding affinities between wild-type Rv2172c, wild-type MSMEG_6649, and its site-directed mutants with NADH or compound AB131 or compound 13 or 14 , the MST binding assays were carried out as described previously. 34 Briefly, the NT- 647-NHS dye from the Monolith NTTM Protein Labeling Kit RED-NHS was first used to label the wild-type Rv2172c, wild-type MSMEG_6649, and their mutants by following the manufacturer’s instructions (NanoTemper Technologies). The labeled Rv2172c, MSMEG_6649, or their mutants were mixed with 1:2 serial dilutions of NADH or AB131 or compound 13 or 14 in PBS buffer (pH 7.8) with 0.05% Tween-20 and incubated for 15 min at 25 °C and then loaded into NT.115 premium-coated capillaries from NanoTemper Technologies.…”

Investigations into the antibacterial effects and potential mechanism of gambogic acid and neogambogic acid

“…To characterize the binding affinity between Efa UPPS and GA or NGA, the MST binding assay was performed as previously described ( Lin et al, 2020 ). In brief, His-tagged Efa UPPS and site-directed mutated Efa UPPS were labeled with the NT- 647-NHS dye using the Monolith NTTM Protein Labeling Kit RED-NHS (NanoTemper Technologies).…”