Department of Structural and Chemical Biology, Mount Sinai School of Medicine, Box 1677, 1425 Madison Avenue, NY 10029, USA.
Nucleic Acids Res. 2011 Sep 1;39(16):7300-7. doi: 10.1093/nar/gkr325. Epub 2011 May 19.
Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-β (IFN-β) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-β promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.
干扰素调节因子 IRF-3 和 IRF-7 是病毒感染时激活干扰素-β (IFN-β) 基因所必需的转录因子。虽然这两种蛋白质都识别相同的 IRF 结合基序 AANNGAAA，但它们在体内对不同的 DNA 结合位点具有不同的 DNA 结合偏好。IRF-3 和 IRF-7 DNA 结合结构域 (DBD) 与 IFN-β 启动子元件结合的 X 射线结构揭示了环 (L1-L3) 和与靶序列相互作用的残基的灵活性。为了表征 DNA 结合过程中发生的构象变化以及它们在 IRF 家族成员之间的差异，我们已经解决了无 DNA 存在时的 IRF-3 和 IRF-7 DBD 的 X 射线结构。我们发现，携带与 DNA 小沟相互作用的保守组氨酸的环 L1 在 apo IRF-3 中无序，但在 apo IRF-7 中有序。这反映在当两个蛋白质中环 L1 中的保守组氨酸突变为丙氨酸时，DNA 结合亲和力的差异。IRF-7 中环 L1 的稳定性源自于一组独特的疏水性残基，它们与蛋白质核心相对。总之，我们的数据表明，环 L1 灵活性的差异是 IRF-DNA 结合差异的一个重要决定因素。
