Targeting MarA N‐terminal domain dynamics to prevent DNA binding

Efflux is one of the mechanisms employed by Gram‐negative bacteria to become resistant to routinely used antibiotics. The inhibition of efflux by targeting their regulators is a promising strategy to re‐sensitize bacterial pathogens to antibiotics. AcrAB–TolC is the main resistance‐nodulation‐division efflux pump in Enterobacteriaceae. MarA is an AraC/XylS family global regulator that regulates more than 40 genes related to the antimicrobial resistance phenotype, including acrAB. The aim of this work was to understand the role of the N‐terminal helix of MarA in the mechanism of DNA binding. An N‐terminal deletion of MarA showed that the N‐terminal helix is critical for recognition of the functional marboxes. By engineering two double cysteine variants of MarA that form a disulfide bond between the N‐terminal helix and the hydrophobic core of one of the helices in direct DNA contact, and combining in vitro electrophoretic mobility assays, in vivo measurements of acrAB transcription using a GFP reporter system, and molecular dynamic simulations, it was shown that the immobilization of the N‐terminal helix of MarA prevents binding to DNA. This inhibited conformation seems to be universal for the monomeric members of the AraC/XylS family, as suggested by additional molecular dynamics simulations of the two‐domain protein Rob. These results point to the N‐terminal helix of the AraC/XylS family monomeric regulators as a promising target for the development of inhibitors.

• Publication year

  2024

• Venue

  Protein Science

• Publication date

  2024-12-11

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1002/pro.5258PMID 39660948PMCID 11633057
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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