Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas, United States of America.
Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, Sealy Center for Vaccine Development, University of Texas Medical Branch, Galveston, Texas, United States of America.
PLoS One. 2018 Apr 11;13(4):e0194891. doi: 10.1371/journal.pone.0194891. eCollection 2018.
Ehrlichia chaffeensis, the causative agent of human monocytotropic ehrlichiosis, secretes several effector proteins that bind host DNA to modulate host gene expression. The tandem repeat protein 120 (TRP120), one of the largest effector proteins, has four nearly identical tandem repeat (TR) regions that each consists of 80 amino acids. In addition to playing a role in ehrlichial binding and internalization, TRP120 translocates to the host nucleus where it is thought to function as a transcription factor that modulates gene expression. However, sequence analysis of TRP120 does not identify the presence of DNA-binding or trans-activation domains typical of classical eukaryotic transcription factors. Thus, the mechanism by which TRP120 binds DNA and modulates gene expression remains elusive. Herein, we expressed the TR regions of the TRP120 protein, and characterized its solution structure and ability to bind DNA. TRP120, expressed as either a one or two TR repeat, is a monomer in solution, and is mostly disordered as determined by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Using NMR spectroscopy, we further show that the 1 TR construct selectively binds GC-rich DNA. Although low pH was required for TRP120 TR-DNA interaction, acidic pH alone does not induce any significant structural changes in the TR region. This suggests that TRP120 folds into an ordered structure upon forming a protein-DNA complex, and thus folding of TRP120 TR is coupled with DNA binding.
查菲埃立克体(Ehrlichia chaffeensis)是人类单核细胞埃立克体病的病原体，它分泌几种效应蛋白，与宿主 DNA 结合，调节宿主基因表达。串联重复蛋白 120(TRP120)是最大的效应蛋白之一，它有四个几乎相同的串联重复(TR)区，每个区由 80 个氨基酸组成。除了在埃立克体的结合和内化中发挥作用外，TRP120 还易位到宿主核内，被认为作为一种转录因子调节基因表达。然而，TRP120 的序列分析并未识别出与经典真核转录因子相似的 DNA 结合或转录激活结构域。因此，TRP120 结合 DNA 和调节基因表达的机制仍不清楚。在此，我们表达了 TRP120 蛋白的 TR 区，并对其溶液结构和结合 DNA 的能力进行了表征。TRP120 无论是表达为一个还是两个 TR 重复，在溶液中都是单体，通过圆二色性(CD)和核磁共振(NMR)光谱确定其大部分是无规卷曲。通过 NMR 光谱，我们进一步表明，1 TR 结构选择性地结合富含 GC 的 DNA。尽管 TRP120 TR-DNA 相互作用需要低 pH 值，但单独的酸性 pH 值不会引起 TR 区发生任何显著的结构变化。这表明 TRP120 在形成蛋白-DNA 复合物时折叠成一个有序的结构，因此 TRP120 TR 的折叠与 DNA 结合相偶联。
