Abstract
ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes. Its versatile functionality as activator, repressor or coactivator suggests a complex regulatory mechanism, whose clarification is essential for a better understanding of the virulence expression system of V. cholerae. Here, we provide structural information elucidating the organization and binding behavior of the cytoplasmic DNA-binding domain of ToxR (cToxR), containing a winged helix–turn–helix (wHTH) motif. Our analysis reveals unexpected structural features of this domain expanding our knowledge of a poorly defined subfamily of wHTH proteins. cToxR forms an extraordinary long α-loop and furthermore has an additional C-terminal beta strand, contacting the N-terminus and thus leading to a compact fold. The identification of the exact interactions between ToxR and DNA contributes to a deeper understanding of this regulatory process. Our findings not only show general binding of the soluble cytoplasmic domain of ToxR to DNA, but also indicate a higher affinity for the toxT motif. These results support the current theory of ToxR being a “DNA-catcher” to enable binding of the transcription factor TcpP and thus activation of virulence-associated toxT transcription. Although, TcpP and ToxR interaction is assumed to be crucial in the activation of the toxT genes, we could not detect an interaction event of their isolated cytoplasmic domains. We therefore conclude that other factors are needed to establish this protein–protein interaction, e.g., membrane attachment, the presence of their full-length proteins and/or other intermediary proteins that may facilitate binding.
Highlights
ToxR represents an essential transcription factor of Vibrio cholerae, which is involved in the regulation of multiple, mainly virulence associated genes
The main virulence factors in V. cholerae are the toxin coregulated pilus (TCP), which is required for the adherence of the bacterium in the small intestine [4] and the cholera toxin (CT), causing the main symptom of the disease, the fatal diarrhea [4, 5]
Because of the high structural similarity between cytoplasmic domain of ToxR (cToxR) and winged helix–turn– helix (wHTH) protein cYycF [37] (PDB code: 2d1v), we decided to use a combination of NMR assignments (Table S1), including structural important NOEs (Table S2), chemical shiftbased secondary structure predictions [40] (Fig. S3), and CS-Rosetta [41, 42]
Summary
Nina Gubensäk1,2,* , Evelyne Schrank, Christoph Hartlmüller, Christoph Göbl , Fabio S. Helge Meyer1,8 , Tea Pavkov-Keller2,9,10 , Tobias Madl, Joachim Reidl, and Klaus Zangger1,9,10,* From the 1Institute of Chemistry/Organic and Bioorganic Chemistry, 2Institute of Molecular Biosciences, University of Graz, Graz, Austria; 3Center for Integrated Protein Science Munich (CIPSM) at the Department of Chemistry, Technische Universität München, Garching, Germany; 4Department of Pathology and Biomedical Science, University of Otago Christchurch, Christchurch, New Zealand; 5Institute of Pharmaceutical Sciences/Pharmaceutical Technology and Biopharmacy, University of Graz, Graz, Austria; 6Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solna, Sweden; 7Diagnostic and Research Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, Graz, Austria; 8Division of Experimental Allergology and Immunodermatology, University of Oldenburg, Oldenburg, Germany; 9BioTechMed-Graz, Graz, Austria; 10Field of Excellence BioHealth, University of Graz, Graz, Austria; 11Gottfried Schatz Research Center for Cell Signaling, Metabolism and Aging, Institute of Molecular Biology & Biochemistry, Medical University of Graz, Graz, Austria
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