Department of Biology, Indiana University, Bloomington, Indiana, USA.
Center for Structural Genomics of Infectious Diseases, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.
mBio. 2021 Mar 16;12(2):e03287-20. doi: 10.1128/mBio.03287-20.
Sequence-specific DNA-binding domains (DBDs) are conserved in all domains of life. These proteins carry out a variety of cellular functions, and there are a number of distinct structural domains already described that allow for sequence-specific DNA binding, including the ubiquitous helix-turn-helix (HTH) domain. In the facultative pathogen , the chitin sensor ChiS is a transcriptional regulator that is critical for the survival of this organism in its marine reservoir. We recently showed that ChiS contains a cryptic DBD in its C terminus. This domain is not homologous to any known DBD, but it is a conserved domain present in other bacterial proteins. Here, we present the crystal structure of the ChiS DBD at a resolution of 1.28 Å. We find that the ChiS DBD contains an HTH domain that is structurally similar to those found in other DNA-binding proteins, like the LacI repressor. However, one striking difference observed in the ChiS DBD is that the canonical tight turn of the HTH is replaced with an insertion containing a β-sheet, a variant which we term the helix-sheet-helix. Through systematic mutagenesis of all positively charged residues within the ChiS DBD, we show that residues within and proximal to the ChiS helix-sheet-helix are critical for DNA binding. Finally, through phylogenetic analyses we show that the ChiS DBD is found in diverse proteobacterial proteins that exhibit distinct domain architectures. Together, these results suggest that the structure described here represents the prototypical member of the ChiS-family of DBDs. Regulating gene expression is essential in all domains of life. This process is commonly facilitated by the activity of DNA-binding transcription factors. There are diverse structural domains that allow proteins to bind to specific DNA sequences. The structural basis underlying how some proteins bind to DNA, however, remains unclear. Previously, we showed that in the major human pathogen , the transcription factor ChiS directly regulates gene expression through a cryptic DNA-binding domain. This domain lacked homology to any known DNA-binding protein. In the current study, we determined the structure of the ChiS DNA-binding domain (DBD) and found that the ChiS-family DBD is a cryptic variant of the ubiquitous helix-turn-helix (HTH) domain. We further demonstrate that this domain is conserved in diverse proteins that may represent a novel group of transcriptional regulators.
序列特异性 DNA 结合结构域 (DBD) 在所有生命领域都保守存在。这些蛋白质执行多种细胞功能，已经描述了许多不同的结构域，允许序列特异性 DNA 结合，包括普遍存在的螺旋-转角-螺旋 (HTH) 结构域。在兼性病原体中，几丁质传感器 ChiS 是一种转录调节剂，对该生物体在海洋库中的生存至关重要。我们最近表明，ChiS 在其 C 末端含有一个隐藏的 DBD。该结构域与任何已知的 DBD 都没有同源性，但它是其他细菌蛋白中存在的保守结构域。在这里，我们以 1.28Å 的分辨率呈现了 ChiS DBD 的晶体结构。我们发现 ChiS DBD 包含一个 HTH 结构域，该结构域在结构上与其他 DNA 结合蛋白（如 LacI 阻遏物）相似。然而，在 ChiS DBD 中观察到的一个显著差异是，HTH 的经典紧密转角被一个包含β-折叠的插入取代，我们将其称为螺旋- 折叠-螺旋。通过对 ChiS DBD 中所有带正电荷残基的系统诱变，我们表明 ChiS 螺旋- 折叠-螺旋内及其附近的残基对于 DNA 结合至关重要。最后，通过系统发育分析，我们表明 ChiS DBD 存在于不同的变形杆菌蛋白中，这些蛋白表现出不同的结构域架构。总之，这些结果表明，这里描述的结构代表了 ChiS 家族 DBD 的原型成员。
在所有生命领域中，调节基因表达都是必不可少的。该过程通常通过 DNA 结合转录因子的活性来促进。有多种结构域可以使蛋白质结合到特定的 DNA 序列上。然而，一些蛋白质与 DNA 结合的结构基础仍然不清楚。以前，我们表明在主要的人类病原体中，转录因子 ChiS 通过一个隐藏的 DNA 结合结构域直接调节基因表达。该结构域与任何已知的 DNA 结合蛋白都没有同源性。在当前的研究中，我们确定了 ChiS DNA 结合结构域 (DBD) 的结构，并发现 ChiS 家族 DBD 是普遍存在的螺旋-转角-螺旋 (HTH) 结构域的一个隐藏变体。我们进一步证明，该结构域在可能代表一组新的转录调节剂的各种蛋白质中保守存在。
