Mechanism of Ligand Recognition by BmrR, the Multidrug-Responding Transcriptional Regulator:  Mutational Analysis of the Ligand-Binding Site†

The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell. The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic α-helix. Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 [Zheleznova et al. (1999) Cell 96, 353?362]. Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands. We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic α-helix, on the binding affinities of six different BmrR inducers. The E134A substitution abolished the binding of all but one inducer. Mutations of the hydrophobic residues contacting the ligand, and of the α-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution. These results indicate that each inducer forms a unique set of contacts within the binding site. The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR. The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles. The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar hydrophobic cations and, in response, activates the expression of a multidrug transporter which expels them out of the cell. The structure of the inducer-binding domain of BmrR, both free and in complex with one of the inducers, tetraphenylphosphonium (TPP), revealed an unusual internal binding site, covered by an amphipathic α-helix. Upon unfolding of this helix, the TPP molecule penetrates into the core of the protein, where it contacts six hydrophobic residues and forms an electrostatic bond with a buried glutamate, E134 [Zheleznova et al. (1999) Cell 96, 353?362]. Here, a structure-based mutational analysis was used to understand how BmrR interacts with a wide variety of ligands. We determined the effects of alanine substitutions of each of the seven residues interacting with TPP, and mutations within the amphipathic α-helix, on the binding affinities of six different BmrR inducers. The E134A substitution abolished the binding of all but one inducer. Mutations of the hydrophobic residues contacting the ligand, and of the α-helix, had more moderate effects, often with the affinity for some inducers increasing and others decreasing as a result of the same substitution. These results indicate that each inducer forms a unique set of contacts within the binding site. The flexible geometry of this site and the lack of involvement of hydrogen bonds in ligand binding are the likely reasons for the extremely broad inducer specificity of BmrR. The similarly broad substrate specificity of multidrug transporters can be governed by the same structural principles.