Ribosome-dependent Vibrio cholerae mRNAse HigB2 is regulated by a β-strand sliding mechanism

Abstract

Toxin–antitoxin (TA) modules are small operons involved in bacterial stress response and persistence. higBA operons form a family of TA modules with an inverted gene organization and a toxin belonging to the RelE/ParE superfamily. Here, we present the crystal structures of chromosomally encoded Vibrio cholerae antitoxin (VcHigA2), toxin (VcHigB2) and their complex, which show significant differences in structure and mechanisms of function compared to the higBA module from plasmid Rts1, the defining member of the family. The VcHigB2 is more closely related to Escherichia coli RelE both in terms of overall structure and the organization of its active site. VcHigB2 is neutralized by VcHigA2, a modular protein with an N-terminal intrinsically disordered toxin-neutralizing segment followed by a C-terminal helix-turn-helix dimerization and DNA binding domain. VcHigA2 binds VcHigB2 with picomolar affinity, which is mainly a consequence of entropically favorable de-solvation of a large hydrophobic binding interface and enthalpically favorable folding of the N-terminal domain into an α-helix followed by a β-strand. This interaction displaces helix α3 of VcHigB2 and at the same time induces a one-residue shift in the register of β-strand β3, thereby flipping the catalytically important Arg64 out of the active site.

Mechanism of regulation and neutralization of the AtaR–AtaT toxin–antitoxin system

“…AtaR intrinsically disordered region neutralizes AtaT. The mechanisms of toxin neutralization by their cognate antitoxin are largely diverse [18][19][20][21][22][23] . AtaR belongs to the ribbon-helix-helix (RHH) family that are typically dimers in solution [24][25][26] .…”

Molecular Characterization of SehB, a Type II Antitoxin of Salmonella enterica Serotype Typhimurium: Amino Acid Residues Involved in DNA-Binding, Homodimerization, Toxin Interaction, and Virulence

“…In solution, SehB was detected forming a dimer, bound or not to DNA containing two palindromic sequences found on the −35 and −10 putative boxes (De la Cruz et al, 2013). A dimer of SehB is required for the transcriptional auto-repression, such as was reported for HigA antitoxin in E. coli (HigAEco), S. flexneri, (HigASfl), V. cholerae (HigAVch), P. aeruginosa (HigAPae), and P. vulgaris (HigAPvu) (Yang et al, 2016;Hadzi et al, 2017;Schureck et al, 2019;Xu et al, 2019;Yoon et al, 2019).…”

Molecular mechanism of two nanobodies that inhibit PAI‐1 activity reveals a modulation at distinct stages of the PAI‐1/plasminogen activator interaction

“…The data were initially phased by molecular replacement using the structure of PAI‐1‐W175F (Protein Data Bank [PDB]: 3Q02) or PAI‐1‐stab (PDB: 1DB2) in active conformation as a search model using PHASER . Nb42 and Nb64 were initially modelled based on homologous Nb structures that were selected using a protein basic local alignment search tool (BLAST) (PDB: 5JA8 and 5JA9, respectively). The structures of the PAI‐1/Nb complexes were improved by iterative rounds of manual rebuilding in Coot and refinement in phenix.refine .…”

“…The obtained diffraction data were processed using autoPROC in default settings, with a high-resolution cutoff on CC 1/2 0.60. 31 The data were initially phased by molecular replacement using the structure of PAI-1-W175F (Protein Data Bank [PDB]: 3Q02 32 ) or PAI-1-stab (PDB: 1DB2 33 protein basic local alignment search tool (BLAST) (PDB: 5JA8 and 5JA9, respectively 35 ). The structures of the PAI-1/Nb complexes were improved by iterative rounds of manual rebuilding in Coot and refinement in phenix.refine.…”