Zheleznova E E, Markham P N, Neyfakh A A, Brennan R G
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland 97201-3098, USA.
Protein Sci. 1997 Nov;6(11):2465-8. doi: 10.1002/pro.5560061122.
In the bacterium Bacillus subtilis, the DNA-binding regulatory protein, BmrR, activates transcription from the multidrug transporter gene, bmr, after binding either rhodamine or tetraphenylphosphonium. These two compounds, which have no structural similarity, are also substrates for the bacterial multidrug transporter. BmrR belongs to the MerR family of transcription activators but differs from the other family members in its ability to bind unrelated small molecule activators. As an initial step in the elucidation of the mechanism by which BmrR recognizes rhodamine and tetraphenylphosphonium and activates transcription, we have crystallized the 144-amino acid-residue carboxy terminal dimerization/ligand-binding domain of the BmrR, named the BRC (BmrR C-terminus). Tetragonal crystals of ligand-free BRC take the space group P4(1)2(1)2, or its enantiomorph P4(3)2(1)2, with unit cell dimensions a = b = 76.3 A, c = 96.0 A, alpha = beta = gamma = 90 degrees. Diffraction is observed to at least 2.7 A resolution at room temperature. In addition, we determined the secondary structure content of ligand-free and rhodamine-bound BRC by circular dichroism. In the ligand-free form, BRC has considerable beta-sheet content (41%) and little alpha-helix structure (13%). After BRC binds rhodamine, its beta-sheet content increases to 47% while the alpha-helix structure decreases to 11%. The structure of BRC will provide insight not only into its multidrug recognition mechanism but could as well aid in the elucidation of the recognition and efflux mechanisms of Bmr and other bacterial multidrug transporters.
在枯草芽孢杆菌中，DNA结合调节蛋白BmrR在与若丹明或四苯基鏻结合后，激活多药转运蛋白基因bmr的转录。这两种化合物没有结构相似性，但也是细菌多药转运蛋白的底物。BmrR属于MerR转录激活因子家族，但在结合不相关的小分子激活剂的能力上与其他家族成员不同。作为阐明BmrR识别若丹明和四苯基鏻并激活转录机制的第一步，我们已使BmrR的144个氨基酸残基的羧基末端二聚化/配体结合结构域（命名为BRC，即BmrR C末端）结晶。无配体BRC的四方晶体属于空间群P4(1)2(1)2或其对映体P4(3)2(1)2，晶胞参数为a = b = 76.3 Å，c = 96.0 Å，α = β = γ = 90°。在室温下观察到衍射分辨率至少为2.7 Å。此外，我们通过圆二色性测定了无配体和结合若丹明的BRC的二级结构含量。在无配体形式下，BRC具有相当多的β-折叠含量（41%）和很少的α-螺旋结构（13%）。BRC与若丹明结合后，其β-折叠含量增加到47%，而α-螺旋结构减少到11%。BRC的结构不仅将有助于深入了解其多药识别机制，还可能有助于阐明Bmr和其他细菌多药转运蛋白的识别和外排机制。
