Targeting MarA N-terminal domain dynamics to prevent DNA binding
Corbella, Marina ; Moreira, Cátia ; Bello-Madruga, Roberto ; Torrent Burgas, Marc ; Kamerlin, Shina C L LU
; Blair, Jessica M A
and Sancho-Vaello, Enea
(2025)
In Protein Science
34(1).
- Abstract
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... (More)
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.
(Less)
- author
- Corbella, Marina
; Moreira, Cátia
; Bello-Madruga, Roberto
; Torrent Burgas, Marc
; Kamerlin, Shina C L
LU
; Blair, Jessica M A
and Sancho-Vaello, Enea
- publishing date
- 2025-01
- type
- Contribution to journal
- publication status
- published
- keywords
- DNA-Binding Proteins/chemistry, Molecular Dynamics Simulation, Escherichia coli/genetics, DNA, Bacterial/metabolism, Protein Binding, Escherichia coli Proteins/chemistry, Protein Domains, Bacterial Proteins/chemistry
- in
- Protein Science
- volume
- 34
- issue
- 1
- article number
- e5258
- pages
- 21 pages
- publisher
- The Protein Society
- external identifiers
-
- scopus:85212146978
- pmid:39660948
- ISSN
- 1469-896X
- DOI
- 10.1002/pro.5258
- language
- English
- LU publication?
- no
- additional info
- © 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
- id
- c4d3a9c3-2446-4942-801e-2556f3f78a16
- date added to LUP
- 2025-01-11 17:57:49
- date last changed
- 2025-07-13 18:37:23
@article{c4d3a9c3-2446-4942-801e-2556f3f78a16,
abstract = {{<p>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 <i>acrAB</i>. 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 <i>acrAB </i>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.</p>}},
author = {{Corbella, Marina and Moreira, Cátia and Bello-Madruga, Roberto and Torrent Burgas, Marc and Kamerlin, Shina C L and Blair, Jessica M A and Sancho-Vaello, Enea}},
issn = {{1469-896X}},
keywords = {{DNA-Binding Proteins/chemistry; Molecular Dynamics Simulation; Escherichia coli/genetics; DNA, Bacterial/metabolism; Protein Binding; Escherichia coli Proteins/chemistry; Protein Domains; Bacterial Proteins/chemistry}},
language = {{eng}},
number = {{1}},
publisher = {{The Protein Society}},
series = {{Protein Science}},
title = {{Targeting MarA N-terminal domain dynamics to prevent DNA binding}},
url = {{http://dx.doi.org/10.1002/pro.5258}},
doi = {{10.1002/pro.5258}},
volume = {{34}},
year = {{2025}},
}