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.

Global Awakening of Cryptic Biosynthetic Gene Clusters in Burkholderia thailandensis

“…14, 15 To confirm this prediction, repression of the mftP-fenI operon by MftR was verified by targeted deletion of mftR followed by determination of transcript levels. As shown in Fig.…”

“…Notably, urate did not result in upregulation of these genes, which were previously seen to be upregulated ~14-fold when cultures were grown with 10 mM urate. 15 The affinity of MftR for each of the two DNA sites in the mftR-mftP intergenic DNA is ~0.7 nM and its affinity for urate is ~6 μM. 15 The Cheng-Prusoff equation 23 relates the IC 50 (the concentration of inhibitor – urate in this case – required for half-maximal inhibition) and affinity for the ligand that is being competed off (DNA).…”

“…1A) is relieved by urate. 14, 15 Since MftR binds urate in vitro , MftR was therefore predicted to repress the divergent genes, with urate acting as the inducer. Urate is the product of xanthine dehydrogenase (XDH), which is also a key enzyme in purine salvage (Fig.…”

Histidine switch controlling pH-dependent protein folding and DNA binding in a transcription factor at the core of synthetic network devices

“…That changes in the dimerization region affect DNA binding has been previously reported for other MarR homologs. 31, 32 This communication between dimerization and DNA-binding regions likely depends on α2 helices, whose C-terminal halves contact the helix-turn-helix motif (Fig. 1A).…”

“…That HucR-H51F exhibited pH-sensitivity may be due to protonation of additional His residues of which one (H25) is located immediately following α1, in the loop connecting α1 and α2, and two are in α6, H142 on the protein surface and H147 facing the ligand-binding pocket that bridges the DNA-binding lobes and the dimer interface. 18, 32 …”