title: Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance.
abstract: The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations-stability, domain interactions, and internal hydrophobic surfaces-are also critical for the regulation of MexR DNA binding.
authors.affiliation: Division of Molecular Biotechnology, Department of Physics, Chemistry and Biology, Linköping University, SE-58183 Linköping, Sweden.
authors.forename: Cecilia
authors.initials: C
authors.lastname: Andrésen
authors.forename: Shah
authors.initials: S
authors.lastname: Jalal
authors.forename: Daniel
authors.initials: D
authors.lastname: Aili
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Sohidul
authors.initials: S
authors.lastname: Islam
authors.forename: Anngelica
authors.initials: A
authors.lastname: Jarl
authors.forename: Bo
authors.initials: B
authors.lastname: Liedberg
authors.forename: Bengt
authors.initials: B
authors.lastname: Wretlind
authors.forename: Lars-Göran
authors.initials: LG
authors.lastname: Mårtensson
authors.forename: Maria
authors.initials: M
authors.lastname: Sunnerhagen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005809:Genes, Regulator / Q000235:genetics*; D009154:Mutation; D009876:Operon; D017510:Protein Folding; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C423620:MexR protein, Pseudomonas aeruginosa; D012097:Repressor Proteins