Structural basis ofBacillus anthracisMoxXT disruption and the modulation of MoxT ribonuclease activity by rationally designed peptides

Abstract

Bacillus anthracis MoxXT is a Type II proteic Toxin-Antitoxin (TA) module wherein MoxT is a ribonuclease that cleaves RNA specifically while MoxX interacts with MoxT and inhibits its activity. Disruption of the TA interaction has been proposed as a novel antibacterial strategy. Peptides, either based on antitoxin sequence or rationally designed, have previously been reported to disrupt the MoxXT interaction but cause a decrease in MoxT ribonuclease activity. In the present study, we report the crystal structure of MoxT, and the effect of several peptides in disrupting the MoxXT interaction as well as augmentation of MoxT ribonuclease activity by binding to MoxT in vitro. Docking studies on the peptides were carried out in order to explain the observed structure activity relationships. The peptides with ribonuclease augmentation activity possess a distinct structure and are proposed to bind to a distinct site on MoxT. The docking of the active peptides with MoxT showed that they possess an aromatic group that occupies a conserved hydrophobic pocket. Additionally, the peptides inducing high ribonuclease activity were anchored by a negatively charged group near a cluster of positively charged residues present near the pocket. Our study provides a structural basis and rationale for the observed properties of the peptides and may aid the development of small molecules to disrupt the TA interaction.

Structural analyses of the MazEF4 toxin-antitoxin pair in Mycobacterium tuberculosis provide evidence for a unique extracellular death factor

“…This region corresponds to an area called Site 1 (Fig. 4), which was named in previous MazF studies (30,33). This agrees well with the E. coli EDF and EDF-like peptide docking studies performed on E. coli MazF and B. anthracis MoxT (26,33).…”

“…4), which was named in previous MazF studies (30,33). This agrees well with the E. coli EDF and EDF-like peptide docking studies performed on E. coli MazF and B. anthracis MoxT (26,33). In the peptide docking model of MoxT, the opening of the S1-S2 loop is required for peptide binding at the bottom of the S1-S2 loop of MoxT, as the area is buried under the S1-S2 loop (33).…”

“…deviation of 1.5 Å for 100 equivalent C␣ positions, Z-score of 16.9, and sequence identity of 29%) (32), and (iv) MoxT from Bacillus anthracis (PDB code 4HKE (chains A and B); r.m.s. deviations of 1.5-1.6 Å for 100 equivalent C␣ positions, Z-scores of 16.6 -16.7, and sequence identity of 27%) (33). Although M. tuberculosis MazF4 shows high Z-scores with its structural homologs, it has a remarkably shorter S1-S2 loop than that of its homologs (Figs.…”

“…The split peaks that correspond to Glu 70 indicate a slightly different conformation of the two monomers constituting MazF4 homodimer in solution. B. anthracis MoxT-EDF models suggest a negatively charged residue of EDF, placed in proximity to Arg 81 and Arg 87 of MoxT, which are structural equivalents of Glu 70 and Glu 76 in MazF4, strongly increases the MoxT activity (33). It is interesting that the arginine residues that are likely to interact with EDF in the structural homologs are replaced with glutamates in MazF4.…”

“…Interestingly, the new EDF homolog from M. tuberculosis we present here has an arginine residue at the fifth position. The positively charged cluster near the S1-S2 loop formed by Arg 81 and Arg 87 in B. anthracis MoxT is required to be occupied by a negatively charged residue for the activity enhancement (33). As the cluster is substituted with negatively charged residues in M. tuberculosis MazF4 (Glu 70 and Glu 76 ), arginine residue at the fifth position is believed to be critical for enhancement of the MazF4 activity.…”