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Modeling of the structure and interactions of the B. anthracis antitoxin, MoxX: deletion mutant studies highlight its modular structure and repressor function

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Abstract

Our previous report on Bacillus anthracis toxin-antitoxin module (MoxXT) identified it to be a two component system wherein, PemK-like toxin (MoxT) functions as a ribonuclease (Agarwal S et al. JBC 285:7254–7270, 2010). The labile antitoxin (MoxX) can bind to/neutralize the action of the toxin and is also a DNA-binding protein mediating autoregulation. In this study, molecular modeling of MoxX in its biologically active dimeric form was done. It was found that it contains a conserved Ribbon-Helix-Helix (RHH) motif, consistent with its DNA-binding function. The modeled MoxX monomers dimerize to form a two-stranded antiparallel ribbon, while the C-terminal region adopts an extended conformation. Knowledge guided protein–protein docking, molecular dynamics simulation, and energy minimization was performed to obtain the structure of the MoxXT complex, which was exploited for the de novo design of a peptide capable of binding to MoxT. It was found that the designed peptide caused a decrease in MoxX binding to MoxT by 42% at a concentration of 2 μM in vitro. We also show that MoxX mediates negative transcriptional autoregulation by binding to its own upstream DNA. The interacting regions of both MoxX and DNA were identified in order to model their complex. The repressor activity of MoxX was found to be mediated by the 16 N-terminal residues that contains the ribbon of the RHH motif. Based on homology with other RHH proteins and deletion mutant studies, we propose a model of the MoxX–DNA interaction, with the antiparallel β-sheet of the MoxX dimer inserted into the major groove of its cognate DNA. The structure of the complex of MoxX with MoxT and its own upstream regulatory region will facilitate design of molecules that can disrupt these interactions, a strategy for development of novel antibacterials.

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Abbreviations

TA:

Toxin-antitoxin

RHH:

Ribbon-helix-helix

ORF:

Open reading frame

CD:

Circular dichroism

aa:

Amino acids

RFI:

Relative fluorescence intensity

MD:

Molecular dynamics

EM:

Energy minimization

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Acknowledgments

Authors would like to acknowledge Prof. B. Jayaram for providing access to Super Computing Facility for Bioinformatics and Computational Biology, IIT (Delhi) for running MD simulations. We also acknowledge the help of Barak Raveh and Ora Schueler-Furman at the Hebrew University, Jerusalem for enabling exhaustive runs of FlexPepDock simulations on their server and for their valuable inputs. N.C. acknowledges University Grants Commission and Council of Scientific and Industrial Research for providing financial assistance.

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Author notes

  1. Shivangi Agarwal

    Present address: Bacterial Pathogenesis Laboratory, Department of Pathology, Tzagournis Medical Research Facility, Ohio State University, Room No: 290, 420 W, 12th Ave, Columbus, OH, 43202, USA

Authors and Affiliations

  1. Division of Biotechnology, Netaji Subhas Institute of Technology, Dwarka, New Delhi, 110078, India

    Nikita Chopra & Sonika Bhatnagar

  2. Laboratory of Molecular Biology and Genetic Engineering, School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India

    Shivangi Agarwal, Shashikala Verma & Rakesh Bhatnagar

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  1. Nikita Chopra
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Corresponding authors

Correspondence to Sonika Bhatnagar or Rakesh Bhatnagar.

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Figure S1

Superposition of MoxXT complex with CcdAB (3HPW). The toxin dimers of the TA complexes were superimposed and are shown here (MoxT in cyan and CcdB in orange). The C-terminus (residues 40–72) of CcdA are shown in yellow while the two chains of MoxX dimer are shown in pink and purple (TIFF 20328 kb)

Supplementary material 2 (DOC 32 kb)

Supplementary material 3 (DOC 27 kb)

Supplementary material 4 (DOC 27 kb)

Supplementary material 5 (DOC 27 kb)

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Chopra, N., Agarwal, S., Verma, S. et al. Modeling of the structure and interactions of the B. anthracis antitoxin, MoxX: deletion mutant studies highlight its modular structure and repressor function. J Comput Aided Mol Des 25, 275–291 (2011). https://doi.org/10.1007/s10822-011-9419-z

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