Binding of the Lactococcal Drug Dependent Transcriptional Regulator LmrR to Its Ligands and Responsive Promoter Regions

Abstract

The heterodimeric ABC transporter LmrCD from Lactococcus lactis is able to extrude several different toxic compounds from the cell, fulfilling a role in the intrinsic and induced drug resistance. The expression of the lmrCD genes is regulated by the multi-drug binding repressor LmrR, which also binds to its own promoter to autoregulate its own expression. Previously, we reported the crystal structure of LmrR in the presence and absence of the drugs Hoechst 33342 and daunomycin. Analysis of the mechanism how drugs control the repressor activity of LmrR is impeded by the fact that these drugs also bind to DNA. Here we identified, using X-ray crystallography and fluorescence, that riboflavin binds into the drug binding cavity of LmrR, adopting a similar binding mode as Hoechst 33342 and daunomycin. Microscale thermophoresis was employed to quantify the binding affinity of LmrR to its responsive promoter regions and to evaluate the cognate site of LmrR in the lmrCD promoter region. Riboflavin reduces the binding affinity of LmrR for the promoter regions. Our results support a model wherein drug binding to LmrR relieves the LmrR dependent repression of the lmrCD genes.

Structural basis of effector and operator recognition by the phenolic acid-responsive transcriptional regulator PadR

“…Thus, a PadR dimer binds two effector molecules with a 1:2 stoichiometry ( Supplementary Figure S15A ). In contrast, LmrR, a member of the PadR subfamily-2, contains only one domain consisting of α1-α4 helices and a β-stranded wing and thus lacks the interdomain pocket observed in PadR ( Supplementary Figures S14A and 15B ) ( 19 , 22 ). Instead, LmrR has exceptionally long terminal helices, α1 and α4, which provide the dimerization interface.…”

“…Despite the discovery of a growing number of PadR family members and the increasing interest in their critical functions, the structural basis of effector-mediated gene regulation is poorly understood in the PadR family because of the highly limited structural information concerning the interactions of PadR family members with effectors and operator DNAs. The structure of LmrR, a subfamily-2 member, in complex with its effectors was solely characterized in bacteria ( 19 , 22 ). LmrR contains a pocket between the two subunits of the LmrR dimer where it binds an effector molecule.…”

Dynamic equilibrium on DNA defines transcriptional regulation of a multidrug binding transcriptional repressor, LmrR

“…S1) 15, 16 . In the previous study, we demonstrated that LmrR exists as a conformational ensemble with multiple α4 helix orientations in solution (Fig.…”

Dynamics of Metal Complex Binding in Relation to Catalytic Activity and Selectivity of an Artificial Metalloenzyme

“…16 This hydrophobic pore serves as a promiscuous binding pocket where planar aromatic molecules bind, as shown in X-ray and NMR structures of LmrR with various planar drugs bound. [16][17][18] Two tryptophan residues, one from each subunit, i.e., W96 and W96', play a key role in binding by sandwiching the guest molecule via π-stacking interactions. Previously, we have shown that this arrangement is attractive for the supramolecular self-assembly of a novel artificial metalloenzyme, by combining the protein LmrR with a Cu(II) complex with a planar aromatic ligand, like 1,10phenanthroline (phen) ( Fig.…”