Structure-based molecular characterization and regulatory mechanism of the LftR transcription factor from Listeria monocytogenes: Conformational flexibilities and a ligand-induced regulatory mechanism

RESEARCH ARTICLE Structure-based molecular characterization and regulatory mechanism of the LftR transcription factor from Listeria monocytogenes: Conformational flexibilities and a ligand-induced regulatory mechanism Choongdeok Lee, Meong Il Kim, Jaewan Park, Minsun Hong ID* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Division of Biological Science and Technology, Yonsei University, Wonju, Republic of Korea * Abstract OPEN ACCESS Citation: Lee C, Kim MI, Park J, Hong M (2019) Structure-based molecular characterization and regulatory mechanism of the LftR transcription factor from Listeria monocytogenes: Conformational flexibilities and a ligand-induced regulatory mechanism. PLoS ONE 14(4): e0215017. https://doi.org/10.1371/journal. pone.0215017 Editor: Finbarr Hayes, University of Manchester, UNITED KINGDOM Received: November 12, 2018 Accepted: March 25, 2019 Published: April 10, 2019 Copyright: © 2019 Lee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: We have deposited the coordinates and structure factors for the LftR structures in the PDB under the following accession numbers: 6ABQ and 6ABT. Funding: This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2015R1D1A1A01057574 and 2018R1A2B6001619 to MH). Listeria monocytogenes is a foodborne pathogen that causes listeriosis and can lead to serious clinical problems, such as sepsis and meningitis, in immunocompromised patients and neonates. Due to a growing number of antibiotic-resistant L. monocytogenes strains, listeriosis can steadily become refractory to antibiotic treatment. To develop novel therapeutics against listeriosis, the drug resistance mechanism of L. monocytogenes needs to be determined. The transcription factor LftR from L. monocytogenes regulates the expression of a putative multidrug resistance transporter, LieAB, and belongs to the PadR-2 subfamily of the PadR family. Despite the functional significance of LftR, our molecular understanding of the transcriptional regulatory mechanism for LftR and even for the PadR-2 subfamily is highly limited. Here, we report the crystal structure of LftR, which forms a dimer and protrudes two winged helix-turn-helix motifs for DNA recognition. Structure-based mutational and comparative analyses showed that LftR interacts with operator DNA through a LftR-specific mode as well as a common mechanism used by the PadR family. Moreover, the LftR dimer harbors one intersubunit cavity in the center of the dimeric structure as a putative ligand-binding site. Finally, conformational flexibilities in the LftR dimer and in the cavity suggest that a ligand-induced regulatory mechanism would be used by the LftR transcription factor. Introduction Listeria monocytogenes is a gram-positive bacillus that is responsible for listeriosis in humans. Listeriosis occurs by gastrointestinal ingestion of food contaminated by L. monocytogenes [1]. L. monocytogenes is able to penetrate the mucosal membranes of the intestine, brain barrier, and placenta. Therefore, L. monocytogenes can cause serious problems, such as sepsis, meningitis, and even death, in elderly and immunocompromised individuals, and may induce PLOS ONE | https://doi.org/10.1371/journal.pone.0215017 April 10, 2019 1 / 15 Structure-based molecular characterization of the LftR transcription factor from Listeria monocytogenes Competing interests: The authors have declared that no competing interests exist. miscarriage or stillbirth in pregnant women [2–6]. In particular, because L. monocytogenes can grow at low temperature, extra caution is required for food storage even during refrigeration [7]. Moreover, since the number of antibiotic-resistant L. monocytogenes strains is increasing [8], listeriosis has steadily become refractory to antibiotic treatment. Therefore, it is necessary to obtain in-depth knowledge about the drug resistance mechanism of L. monocytogenes and to develop novel antibiotics against listeriosis. The lftR gene that encodes a Listeria protein facilitating invasion/transcriptional regulator was first identified in 2015 and is commonly found in diverse L. monocytogenes strains [9]. The LftR protein transcriptionally regulates the expression of the lmo0980 gene, which encodes a putative multidrug resistance transporter, LieAB (Listerial importer of ethidium bromide as artificial substrate), and facilitates the export of toxic chemicals from the cytoplasm to the extracellular space [9]. In addition, deletion of the lftR gene decreases the infection rate in host cells, implicating that LftR is a key virulence factor in L. monocytogenes-mediated pathogenicity. Based on an analysis of primary amino acid sequences, LftR belongs to the phenolic acid decarboxylase regulator (PadR) family. In various bacteria, including Bacillus subtilis, L. monocytogenes, Pediococcus pentosaceus, Lactococcus lactis, and Vibrio cholera [10–14], PadR family members were shown to enhance bacterial survival. In the presence of toxic compounds, such as phenolic acids and antibiotic chemicals, PadR family proteins directly sense toxic substances and transcriptionally upregulate the expression of detoxifying enzymes or efflux pumps to lower the intracellular concentration of detrimental chemicals. As transcriptional regulators, PadR family members commonly contain a winged helix-turn-helix (wHTH) motif to interact with operator DNA. Due to intrinsic variations in sizes and tertiary structures, the PadR family is further divided into two subfamilies, PadR-1 and PadR-2, which consists of ~200 and ~100 residues, respectively. PadR-1 subfamily proteins contain two structurally and functionally separated domains, namely an N-terminal wHTH domain for DNA interaction and a C-terminal helical domain for dimerization. The structural study of a PadR-1 member from B. subtilis (BsPadR) demonstrated that each subunit of the BsPadR dimer accommodates one ligand molecule in an intramolecular pocket between the N-terminal and the C-terminal domains, resulting in a molecular stoichiometry of 2:2 [15]. Unlike the PadR-1 subfamily, the PadR-2 subfamily adopts a single-domain structure and forms a dimer using the helices located before and after the wHTH motif. A member of the PadR-2 subfamily, LmrR from L. lactis, was structurally resolved to accommodate one ligand molecule into an intersubunit pocket of an LmrR dimer [16–18]. The comparative analysis of the BsPadR-DNA and BsPadR-ligand structures demonstrated that a PadR-1 subfamily member mediates transcriptional regulation through ligandinduced allostery [15]. However, our understanding of the transcription regulatory mechanism of the PadR-2 subfamily is hi (...truncated)