ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix-turn-helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a "DNA-catcher" to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein-protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.

中文翻译：

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霍乱弧菌转录因子 ToxR 细胞质域的结构和 DNA 结合特性。

ToxR 是霍乱弧菌必不可少的转录因子，它参与多种基因的调控，主要是毒力相关基因。它作为激活剂、阻遏物或共激活剂的多功能功能表明了一种复杂的调节机制，其澄清对于更好地了解霍乱弧菌的毒力表达系统至关重要。在这里，我们提供了结构信息，阐明了 ToxR (cToxR) 的细胞质 DNA 结合域的组织和结合行为，其中包含有翼螺旋-转角-螺旋 (wHTH) 基序。我们的分析揭示了该域出人意料的结构特征，扩展了我们对一个定义不明确的 wHTH 蛋白亚家族的了解。cToxR 形成了一个非常长的 α 环，此外还有一个额外的 C 端 β 链，接触 N 端，从而导致紧凑的折叠。确定 ToxR 和 DNA 之间的确切相互作用有助于更深入地了解这一监管过程。我们的发现不仅表明 ToxR 的可溶性细胞质结构域与 DNA 的普遍结合，而且还表明对 toxT 基序具有更高的亲和力。这些结果支持 ToxR 是“DNA 捕捉器”的当前理论，能够结合转录因子 TcpP，从而激活毒力相关的 toxT 转录。尽管假设 TcpP 和 ToxR 相互作用对 toxT 基因的激活至关重要，但我们无法检测到它们分离的细胞质结构域的相互作用事件。因此我们得出结论，需要其他因素来建立这种蛋白质 - 蛋白质相互作用，例如膜附着，