Department of Biochemistry and Molecular Biology, Center for Biomolecular Structure and Function, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
J Mol Biol. 2010 Apr 30;398(2):264-75. doi: 10.1016/j.jmb.2010.03.011. Epub 2010 Mar 15.
The multidrug-binding transcription regulator BmrR from Bacillus subtilis is a MerR family member that binds to a wide array of cationic lipophilic toxins to activate the transcription of the multidrug efflux pump gene bmr. Transcription activation from the sigma(A)-dependent bmr operator requires BmrR to remodel the nonoptimal 19-bp spacer between the -10 promoter element and the -35 promoter element in order to facilitate productive RNA polymerase binding. Despite the availability of several structures of BmrR bound to DNA and drugs, the lack of a BmrR structure in its unliganded or apo (DNA free and drug free) state hinders our full understanding of the structural transitions required for DNA binding and transcription activation. Here, we report the crystal structure of the constitutively active, unliganded BmrR mutant BmrR(E253Q/R275E). Superposition of the ligand-free (apo BmrR(E253Q/R275E)) and DNA-bound BmrR structures reveals that apo BmrR must undergo significant rearrangement in order to assume the DNA-bound conformation, including an outward rotation of minor groove binding wings, an inward movement of helix-turn-helix motifs, and a downward relocation of pliable coiled-coil helices. Computational analysis of the DNA-free and DNA-bound structures reveals a flexible joint that is located at the center of the coiled-coil helices. This region, which is composed of residues 94 through 98, overlaps the helical bulge that is observed only in the apo BmrR structure. This conformational hinge is likely common to other MerR family members with large effector-binding domains, but appears to be missing from the smaller metal-binding MerR family members. Interestingly, the center-to-center distance of the recognition helices of apo BmrR is 34 A and suggests that the conformational change from the apo BmrR structure to the bmr operator-bound BmrR structure is initiated by the binding of this transcription activator to a more B-DNA-like conformation.
枯草芽孢杆菌中的多药结合转录调节因子 BmrR 是 MerR 家族的一员，它可以结合多种阳离子亲脂性毒素，激活多药外排泵基因 bmr 的转录。依赖于 sigma(A)的 bmr 操纵子的转录激活需要 BmrR 重塑 -10 启动子元件和 -35 启动子元件之间的非最佳 19 个碱基对间隔区，以促进有功能的 RNA 聚合酶结合。尽管有几个 BmrR 与 DNA 和药物结合的结构，但缺乏无配体或apo（无 DNA 和无药物）状态下的 BmrR 结构，这阻碍了我们对 DNA 结合和转录激活所需的结构转变的全面理解。在这里，我们报告了组成型激活的、无配体的 BmrR 突变体 BmrR(E253Q/R275E)的晶体结构。无配体（apo BmrR(E253Q/R275E)）和 DNA 结合 BmrR 结构的叠加表明，apo BmrR 必须经历显著的重排才能采取 DNA 结合构象，包括小沟结合翼的向外旋转、螺旋-转角-螺旋结构的向内运动以及柔韧卷曲螺旋的向下重新定位。对无 DNA 和 DNA 结合结构的计算分析揭示了一个位于卷曲螺旋中心的灵活连接点。该区域由残基 94 至 98 组成，与仅在 apo BmrR 结构中观察到的螺旋膨出重叠。这个构象铰链可能是其他具有大效应物结合结构域的 MerR 家族成员所共有的，但似乎缺失于较小的金属结合 MerR 家族成员中。有趣的是，apo BmrR 的识别螺旋的中心到中心距离为 34Å，表明从 apo BmrR 结构到 bmr 操纵子结合的 BmrR 结构的构象变化是由转录激活剂与更类似于 B-DNA 的构象结合引发的。
