Crystal Structure of Fad35R from Mycobacterium tuberculosis H37Rv in the Apo-State

Fad35R from Mycobacterium tuberculosis binds to the promoter site of Fad35 operon and its DNA binding activities are reduced in the presence of tetracycline and palmitoyl-CoA. We resolved the crystal structure of Fad35R using single-wavelength anomalous diffraction method (SAD). Fad35R comprises canonical DNA binding domain (DBD) and ligand binding domain (LBD), but displays several distinct structural features. Two recognition helices of two monomers in the homodimer are separated by ~ 48 Å and two core triangle-shaped ligand binding cavities are well exposed to solvent. Structural comparison with DesT and QacR structures suggests that ligand binding-induced movement of α7, which adopts a straight conformation in the Fad35R, may be crucial to switch the conformational states between repressive and derepressive forms. Two DBDs are packed asymmetrically, creating an alternative dimer interface which coincides with the possible tetramer interface that connects the two canonical dimers. Quaternary state of alternative dimer mimics a closed-state structure in which two recognition helices are distanced at ~ 35 Å and ligand binding pockets are inaccessible. Results of biophysical studies indicate that Fad35R has the propensity to oligomerize in solution in the presence of tetracycline. We present the first structure of a FadR homologue from mycobacterium and the structure reveals DNA and ligand binding features of Fad35R and also provides a view on alternative quaternary states that mimic open and closed forms of the regulator.

结核分枝杆菌（Mycobacterium tuberculosis）来源的Fad35R蛋白可结合Fad35操纵子（Fad35 operon）的启动子区域，且其DNA结合活性在四环素与棕榈酰辅酶A（palmitoyl-CoA）存在时会减弱。本研究通过单波长反常衍射法（single-wavelength anomalous diffraction, SAD）解析了Fad35R的晶体结构。Fad35R包含典型的DNA结合结构域（DNA binding domain, DBD）与配体结合结构域（ligand binding domain, LBD），但具备若干独特的结构特征。该同源二聚体中两个单体的两条识别螺旋间距约为48埃（Å），且两个核心三角形配体结合空腔充分暴露于溶剂环境。与DesT及QacR的结构进行比对后发现，Fad35R中原本呈直构象的α7螺旋在配体结合后发生移动，这一过程可能是调控阻遏与去阻遏构象转换的关键。两个DNA结合结构域呈不对称排布，形成了一种替代型二聚体界面，该界面与连接两个典型二聚体的潜在四聚体界面相吻合。这种替代型二聚体的四级结构模拟了闭合态构象：此时两条识别螺旋间距约为35埃，配体结合口袋处于不可达状态。生物物理实验结果显示，Fad35R在四环素存在的溶液环境中具有寡聚化倾向。本研究首次解析了分枝杆菌来源的FadR同源蛋白的晶体结构，该结构不仅揭示了Fad35R的DNA与配体结合特征，还展现了该调控蛋白模拟开放与闭合构象的替代型四级结构状态。