The anti-toxin ParD of plasmid RK2 consists of two structurally distinct moieties and belongs to the ribbon-helix-helix family of DNA-binding proteins

Abstract

NMR and CD spectroscopy have been used to characterize, both structurally and dynamically, the 82-amino-acid ParD protein of the post-segregational killing module of the broad-host-range plasmid RP4/RK2. ParD occurs as a dimer in solution and exercises two different control functions; an autoregulatory function by binding to its own promoter P(parDE) and a plasmid-stabilizing function by inhibiting ParE toxicity in cells that express ParD and ParE. Analysis of the secondary structure based on the chemical-shift indices, sequential nuclear Overhauser enhancements (NOEs) and (3)J(Halpha-NH) scalar coupling constants showed that the N-terminal domain of ParD consists of a short beta-ribbon followed by three alpha-helices, demonstrating that ParD contains a ribbon-helix-helix fold, a DNA-binding motif found in a family of small prokaryotic repressors. (15)N longitudinal (T(1)) and transverse (T(2)) relaxation measurements and hetero nuclear NOEs showed that ParD is divided into two separate domains, a well-ordered N-terminal domain and a very flexible C-terminal domain. An increase in secondary structure was observed upon addition of trifluoroethanol, suggested to result from the formation of structured stretches in the C-terminal part of the protein. This is the first experimental evidence that the DNA-binding domain of ParD belongs to the ribbon-helix-helix fold family, and this structural motif is proposed to be present in functionally similar antidote proteins.

ParG, a protein required for active partition of bacterial plasmids, has a dimeric ribbon–helix–helix structure

“…The NMR structure calculations confirmed that ParG belongs to the MetJ/Arc structural superfamily, which currently consists of transcriptional repressors Met (Rafferty et al ., 1989; Somers and Phillips, 1992), Arc (Breg et al ., 1990; Raumann et al ., 1994; Schildbach et al ., 1995, 1999; Waldburger et al ., 1995), CopG (Gomis‐Rüth et al ., 1998), Mnt (Burgering et al ., 1994) and protein ω (Murayama et al ., 2001). It was recently suggested that ParD (Oberer et al ., 2002), NikR (Chivers and Sauer, 1999), TraY (Lum et al . 2002), 7k M k (Pavlov et al ., 2002) and a number of CopG‐like proteins encoded by plasmids of the pMV158 family (del Solar et al ., 2002) will also share a similar RHH fold.…”

“…The alignment illustrates that although different members of this family share the same RHH motif which forms an intertwined dimer, the main difference between them lies in the additional N‐ and/or C‐terminal domains – whether these are present and their lengths. It appears that while the RHH is involved in DNA binding (a common feature of transcriptional repressors), the additional N‐ or C‐terminal regions, when present, are responsible for additional functions: for example, the C‐terminal region of the antitoxin ParD which is flexible in solution (Oberer et al ., 2002) is responsible for binding with a toxin protein, ParE (Roberts et al ., 1993), and the C‐terminal region of MetJ is responsible for co‐repressor binding (Rafferty et al ., 1989). In many other cases, however, the roles of these additional regions remain to be established.…”

Recovery of Plasmid pEMB1, Whose Toxin-Antitoxin System Stabilizes an Ampicillin Resistance-Conferring β-Lactamase Gene in Escherichia coli, from Natural Environments

“…The predicted protein product of orf5 showed 92 and 79% identities to a hypothetical protein PAAH01 (predicted as a transcriptional regulator) from Aeromonas hydrophila (YP_005230994.1) and to the CopG family transcriptional regulator from Shewanella putrefaciens 200 (YP_006010550.1), respectively. The translated orf5 encoded a 79-amino-acid protein that included a ribbonhelix-helix (RHH) structure from the RHH family of proteins, which includes an antitoxin ParD and transcriptional repressors CopG, Arc, and Mnt (38), although their sequence identities were very low (Fig. 3).…”

The solution structure of ParD, the antidote of the ParDE toxin–antitoxin module, provides the structural basis for DNA and toxin binding

“…In contrast to other TA systems that require the complex for full negative regulation of the operon, ParD alone is sufficient for autorepression (Davis et al 1992; Eberl et al 1992). Our previous studies on ParD showed that the protein exists as a dimer in solution, and that ParD exhibits high thermal stability and excellent refolding properties after heat‐induced denaturation (Oberer et al 1999, 2002). Circular dichroism spectroscopy and preliminary characterization from NMR data indicated that the protein was composed of α‐helical and β‐strand regions.…”

“…Circular dichroism spectroscopy and preliminary characterization from NMR data indicated that the protein was composed of α‐helical and β‐strand regions. Chemical shift analysis as well as relaxation data revealed that ParD consists of two structurally distinct moieties, namely a well‐ordered N‐terminal half and an unstructured C‐terminal half (Oberer et al 2002). Among other TA systems, mutational and structural studies confirm the suggested separation into an N‐terminal region, which is mainly responsible for autoregulation, and a C‐terminal region, which functions in neutralization of the toxin (e.g., ParD/Pem [Ruiz‐Echevarria et al 1991a,b; Santos‐Sierra et al 2002], CcdA [Bernard and Couturier 1991; Salmon et al 1994; Madl et al 2006], Phd [Lehnherr et al 1993; Smith and Magnuson 2004; McKinley and Magnuson 2005], MazE 2 MazF 4 [Kamada et al 2003], RelE 2 ‐RelB 2 [Takagi et al 2005], and YefM2‐YoeB [Kamada and Hanaoka 2005]).…”