Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain.

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

TRP120-1TR selectively binds to GC-rich DNA.

<p>(A-B) HSQC spectra of ¹⁵N-labeled TRP120-1TR with GC-rich DNA at pH 7.0 or pH 5.5. TRP120-1TR (68 μM) alone (black) and with a 10-fold molar equivalent of unlabeled DNA (red, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194891#pone.0194891.t001" target="_blank">Table 1</a>) in 20 mM Tris pH 7.0 containing 100 mM NaCl or in 10 mM phosphate pH 5.5 containing 100 mM NaCl are shown. The lines highlight Asn or Gln side chain NH₂ peaks that show distinctly different chemical shifts in the bound form. (C-D) HSQC spectra of ¹⁵N-labeled TRP120-1TR with AT-rich DNA probe at pH 7.0 or pH 5.5. TRP120-1TR (30 μM) alone (black) and with a 10-fold molar equivalent of unlabeled DNA (blue, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194891#pone.0194891.t001" target="_blank">Table 1</a>) in 20 mM Tris pH 7.0 containing 100 mM NaCl or in 10 mM phosphate pH 5.5 containing 100 mM NaCl are shown.</p

DNA probes used in NMR titrations.

<p>DNA probes used in NMR titrations.</p

Sedimentation velocity profiles of TRP120-1TR and TRP120-2TR proteins.

<p>The molecular weights of TRP120-1TR and -2TR proteins were determined by sedimentation velocity experiments. Sedimentation coefficient distribution <i>c(s)</i> profile for TRP120-1TR and TRP120-2TR show one major species, which corresponds to the molecular weight of 12.3 or 22.2 kDa, respectively. The observed molecular weights indicate that both TRP120 constructs are monomers in solution.</p