Ligand-binding pocket bridges DNA-binding and dimerization domains of the urate-responsive MarR homologue MftR from Burkholderia thailandensis

Abstract

Members of the multiple antibiotic resistance regulator (MarR) family often regulate gene activity by responding to a specific ligand. In the absence of ligand, most MarR proteins function as repressors, while ligand binding causes attenuated DNA binding and therefore increased gene expression. Previously, we have shown that urate is a ligand for MftR (major facilitator transport regulator), which is encoded by the soil bacterium Burkholderia thailandensis. We show here that both mftR and the divergently oriented gene mftP encoding a major facilitator transport protein are upregulated in the presence of urate. MftR binds two cognate sites in the mftR-mftP intergenic region with equivalent affinity and sensitivity to urate. Mutagenesis of four conserved residues previously reported to be involved in urate binding to Deinococcus radiodurans HucR and Rhizobium radiobacter PecS significantly reduced protein stability and DNA binding affinity but not ligand binding. These data suggest that residues equivalent to those implicated in ligand binding to HucR and PecS serve structural roles and that MftR relies on distinct residues for ligand binding. MftR exhibits a two-step melting transition suggesting independent unfolding of the dimerization and DNA-binding regions; urate binding or mutations in the predicted ligand-binding sites result in one-step unfolding transitions. We suggest that MftR binds the ligand in a cleft between the DNA-binding lobes and the dimer interface but that the mechanism of ligand-mediated attenuation of DNA binding differs from that proposed for other urate-responsive MarR homologues. Since DNA binding by MftR is attenuated at 37 °C, our data also suggest that MftR responds to both ligand and a thermal upshift by attenuated DNA binding and upregulation of the genes under its control.

Figure 1

B. thailandensismftR-mftP intergenic region. Genes are represented by arrows. The sequence of the intergenic region, mftO, is shown with two imperfect palindromes shown in bold face. mftrO represents a DNA construct with the palindrome near mftR, and mftpO is the region upstream of mftP.

Figure 2

Sequence alignment of MarR homologues. (A) Alignment generated using MUSCLE. Residues involved in urate binding or in communicating its binding to the recognition helix are shown using arrows. The alignment includes MTH313 (Methanobacterium thermoautotrophicum), D. radiodurans HucR, S. coelicolor PecS, D. dadantii PecS, Pectobacterium atrosepticum PecS, R. radiobacter PecS, MftR from B. thailandensis, B. mallei (BMA_0906), and B. pseudomallei (BURPS_1958). Secondary structure elements are based on the structure of HucR. (B) Phylogenetic tree of selected MarR homologues based on their amino acid sequence. Tree includes MarR homologues from panel A and B. oklahomensis and Rhizobium mesoamericanum PecS. The evolutionary distances are in units of the number of amino acid substitutions per position, and the scale bar represents an evolutionary distance of 0.1.

Figure 3

Predicted model of MftR. MftR model based on the structure of HucR (2fbk), created using SwissModel in automated mode. One monomer is colored blue to red (amino-terminus to carboxy-terminus; helices are shown as α1 to α7) and the other is in purple. Conserved residues, which are predicted to bind urate, are in red stick representation.

Figure 4

In vivo transcript level of mftR and mftP. (A) PCR product (mftP) obtained from cDNA generated with increasing concentration of total RNA extracted from cells not exposed to exogenous urate. Lane M is the 100 bp marker, and lanes 1 to 5 show the PCR product obtained with increasing concentrations of RNA (25–100 ng/μL). (B) PCR product (mftP) with RNA extracted from cells grown in the presence of 10 mM urate. Lane M is the 100 bp marker and lanes 1 to 5 show the PCR product obtained with increasing concentrations of RNA (25–100 ng/μL). (C) Relative abundance of mftR and mftP transcript levels after the addition of 10 mM urate. Relative abundance of transcript level was calculated with the comparative C_T method, with the reference control gene gapdh. Error bars represent the standard deviation of three experiments.

Figure 5

MftR binds both palindromes in its operator DNA, and the complexes are sensitive to urate. (A) EMSA showing mftpO (3.0 nM) titrated with increasing concentrations of MftR (0.1–200 nM; lanes 2–15); reaction in lane 1 contains DNA only. Complex and free DNA are identified at the right as C and F, respectively. (B) Fractional complex formation plotted as a function of MftR concentration. Binding isotherm with mftrO (○; solid line) and mftpO (+; dashed line). (C) Effect of urate on the binding of MftR to mftpO. Lane 1 contains DNA only. Reaction in lane 2 contains no ligand. The MftR-mftpO complex was titrated with increasing concentrations of urate (3–18 mM; lanes 3–11). (D) Normalized complex fraction as a function of urate concentration. MftR-mftrO complex (○; solid line) and MftR-mftpO complex (+; dashed line) titrated with increasing concentrations of urate. Error bars represent the standard deviation of three independent repeats.

Figure 6

Melting temperature of MftR and mutant proteins determined by differential scanning fluorometry. Thermal denaturation is represented by the fluorescence intensity resulting from the binding of SYPRO Orange to denatured protein as a function of temperature. (A) Thermal denaturation profile of WT MftR. (B) Thermal denaturation profile of D56S, R63S, and R89N. (C) Melting profile of W11F; a higher initial fluorescence may reflect the presence of already denatured protein. (D) Melting profile of WT with the addition of 100 μM urate. (E) Denaturation profile of D56S, R63S, and R89N with urate. (F) Denaturation profile of W11F with urate. (G) Thermal denaturation profile of MftR with 6 μM mftpO. (H) Denaturation profile of mutant D56S, R63S, and R89N with mftpO. (I) Symbols used in panels A–H for MftR and mutants.

Figure 7

Fluorescence of MftR and mutant proteins and response to urate. (A) Fluorescence profiles of MftR mutants. Mutation of W11 to F (solid black dashed line) causes a significant loss in the intrinsic fluorescence. WT MftR (●), D56S (×), R63S (solid gray line), and R89N (◊). (B) Fluorescence quenching upon urate addition. WT MftR profile is shown as a solid black line and open square with ×. W11F (+) profile with dashed line, and D56S (⧫) with gray lines.