Ehrlichia chaffeensis TRP120 nucleomodulin binds DNA with disordered tandem repeat domain

Abstract

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.

Brucella abortus BspJ Is a Nucleomodulin That Inhibits Macrophage Apoptosis and Promotes Intracellular Survival of Brucella

“…TRP120, another effector of Ehrlichia chaffeensis, not only plays a role in protein binding and internalization but also translocates to the host cell nucleus, and it is thus considered to be a transcription factor that regulates gene expression. However, the mechanism by which TRP120 binds to DNA and regulates gene expression is still elusive (Klema et al, 2018). In general, although we have discovered the effector protein BspJ of Brucella in the nucleus consistent with reported earlier (Myeni et al, 2013), the mechanism of its entry into the nucleus is still unknown, and the effector function after entering the nucleus is worth exploring.…”

Ehrlichia chaffeensis TRP47 enters the nucleus via a MYND-binding domain-dependent mechanism and predominantly binds enhancers of host genes associated with signal transduction, cytoskeletal organization, and immune response

“…This phenomenon has been previously described for several transcription factors [ 33 , 34 ] and is likely due to competition between the antibody and DNA binding of the TRP47 tandem repeat region since the antibody used was directed against a molecularly defined epitope in the tandem repeat domain. Interestingly, a recent study using nuclear magnetic resonance spectroscopy found that TRP120-1TR construct specifically binds DNA only at low pH and with affinity in the micromolar range weaker than that of zinc finger transcription factors [ 35 ]. Further, TRPs may require a post-translational modification or interaction with host or ehrlichial proteins to adopt an ideal conformation for DNA binding, neither of which can occur in the in vitro EMSA assay.…”

Bacterial Factors Targeting the Nucleus: The Growing Family of Nucleomodulins

“…Overlap in the function of target genes could be a strategy developed by E. chaffeensis to secure the modulation of certain cellular signaling during infection. The mechanism by which ehrlichial nucleomodulins bind to DNA involves disordered tandem repeats that fold into an ordered structure when bound to DNA [50]. In addition to its binding to DNA, TRP120 interacts with several epifactors, including the RING domain of PCGF5 [51], a component of a PRC1-like complex.…”