The E. coli cytidine repressor (CytR) is a member of the LacR family of bacterial repressors that regulates nine operons with distinct spacing and orientations of recognition sites. Understanding the structural features of the CytR DNA-binding domain (DBD) when bound to DNA is critical to understanding differential mechanisms of gene regulation. We previously reported the structure of the CytR DBD monomer bound specifically to half-site DNA and found that the DBD exists as a three-helix bundle containing a canonical helix-turn-helix motif, similar to other proteins that interact with DNA [Moody, et al (2011), Biochemistry 50:6622-32]. We also studied the free state of the monomer and found that since NMR spectra show it populates up to four distinct conformations, the free state exists as an intrinsically disordered protein (IDP). Here, we present further analysis of the DBD structure and dynamics in the context of full-site operator or nonspecific DNA. DBDs bound to full-site DNA show one set of NMR signals, consistent with fast exchange between the two binding sites. When bound to full-length DNA, we observed only slight changes in structure compared to the monomer structure and no folding of the hinge helix. Notably, the CytR DBD behaves quite differently when bound to nonspecific DNA compared to LacR. A dearth of NOEs and complete lack of protection from hydrogen exchange are consistent with the protein populating a flexible, molten state when associated with DNA nonspecifically, similar to fuzzy complexes. The CytR DBD structure is significantly more stable when bound specifically to the udp half-site substrate. For CytR, the transition from nonspecific association to specific recognition results in substantial changes in protein mobility that are coupled to structural rearrangements. These effects are more pronounced in the CytR DBD compared to other LacR family members.

中文翻译：

---

胞苷阻遏物DNA结合域内的特异性的动态后果。

大肠杆菌胞苷阻遏物（CytR）是LacR细菌阻遏物家族的成员，该家族以识别位点的不同间距和方向调节九个操纵子。当与DNA结合时，了解CytR DNA结合结构域（DBD）的结构特征对于理解基因调控的不同机制至关重要。我们先前报道了CytR DBD单体与半位点DNA特异性结合的结构，并发现DBD以三螺旋束的形式存在，包含标准的螺旋-转-螺旋基序，类似于与DNA相互作用的其他蛋白质[Moody，等（2011），生物化学50：6622-32]。我们还研究了单体的游离态，发现由于NMR谱图显示该单体存在多达四个不同的构象，因此游离态以固有的无序蛋白（IDP）的形式存在。这里，我们提出了在全位操作员或非特异性DNA的背景下对DBD结构和动力学的进一步分析。绑定到全位DNA的DBD显示一组NMR信号，这与两个结合位点之间的快速交换一致。当与全长DNA结合时，与单体结构相比，我们仅观察到结构上的微小变化，而铰链螺旋没有折叠。值得注意的是，当与非特异性DNA结合时，与LacR相比，CytR DBD的行为有很大不同。当与DNA非特异性结合时，类似于模糊复合物，NOE的缺乏和对氢交换的完全缺乏保护与这种蛋白质在柔性，熔融状态下形成了一致。当与udp半位底物特异性结合时，CytR DBD结构明显更稳定。对于CytR，从非特异性结合到特异性识别的转变会导致蛋白质迁移率发生实质性变化，从而导致结构重排。与其他LacR家族成员相比，这些效应在CytR DBD中更为明显。