Targeting MarA N-terminal domain dynamics to prevent DNA binding

Area of Science:

• Microbiology
• Molecular Biology
• Biochemistry

Background:

• Gram-negative bacteria utilize efflux pumps, like AcrAB-TolC, as a key mechanism for antibiotic resistance.
• MarA, a global regulator from the AraC/XylS family, controls numerous antimicrobial resistance genes, including acrAB, in Enterobacteriaceae.

Purpose of the Study:

• To elucidate the specific role of the N-terminal helix of the MarA regulator in its DNA-binding mechanism.
• To investigate the potential of targeting the MarA N-terminal helix for developing novel strategies against antibiotic resistance.

Main Methods:

• Constructing MarA variants with engineered disulfide bonds to immobilize the N-terminal helix.
• Employing in vitro electrophoretic mobility assays and in vivo reporter systems to assess DNA binding and gene transcription.
• Utilizing molecular dynamic simulations to analyze the conformational effects of N-terminal helix immobilization on MarA and related proteins.

Main Results:

• Deletion of the N-terminal helix demonstrated its essentiality for recognizing functional marboxes, critical DNA binding sites.
• Immobilization of the N-terminal helix via disulfide bonds abolished MarA's ability to bind DNA.
• Molecular dynamics simulations suggested this inhibitory conformation is conserved among monomeric AraC/XylS family regulators.

Conclusions:

• The N-terminal helix of MarA is indispensable for DNA binding and subsequent regulation of antibiotic resistance genes.
• The N-terminal helix of monomeric AraC/XylS regulators represents a potential therapeutic target for combating bacterial antibiotic resistance.