N. Vazquez-laslop et al., Mechanism of ligand recognition by BmrR, the multidrug-responding transcriptional regulator: Mutational analysis of the ligand-binding site, BIOCHEM, 38(51), 1999, pp. 16925-16931
The Bacillus subtilis transcriptional regulator BmrR recognizes dissimilar
hydrophobic cations and, in response, activates the expression of a multidr
ug transporter which expels them out of the cell. The structure of the indu
cer-binding domain of BmrR, both free and in complex with one of the induce
rs, tetraphenylphosphonium (TPP), revealed an unusual internal binding site
, covered by an amphipathic alpha-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 glutama
te, E134 [Zheleznova et al. (1999) Cell 96, 353-362]. Here, a structure-bas
ed mutational analysis was used to understand how BmrR interacts with a wid
e variety of ligands. We determined the effects of alanine substitutions of
each of the seven residues interacting with TPP, and mutations within the
amphipathic alpha-helix. on the binding affinities of six different BmrR in
ducers. The E134A substitution abolished the binding of all but one inducer
. Mutations of the hydrophobic residues contacting the ligand, and of the a
lpha-helix, had more moderate effects, often with the affinity for some ind
ucers increasing and others decreasing as a result of the same substitution
. These results indicate that each inducer forms a unique set of contacts w
ithin 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 substr
ate specificity of multidrug transporters can be governed by the same struc
tural principles.