The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains
Margot Di Cesare
(1)
,
Elise Kaplan
(1)
,
Julia Rendon
(2)
,
Guillaume Gerbaud
(2)
,
Sepideh Valimehr
(3)
,
Alexia Gobet
(1)
,
Thu-Anh Thi Ngo
(1)
,
Vincent Chaptal
(1)
,
Pierre Falson
(1)
,
Marlène Martinho
(2)
,
Pierre Dorlet
(4)
,
Eric Hanssen
(3)
,
Jean-Michel Jault
(1)
,
Cédric Orelle
(1)
Margot Di Cesare
- Fonction : Auteur
- PersonId : 821824
- ORCID : 0000-0002-7883-852X
Elise Kaplan
- Fonction : Auteur
- PersonId : 1346373
- ORCID : 0000-0003-4985-1482
Guillaume Gerbaud
- Fonction : Auteur
- PersonId : 779947
- ORCID : 0000-0002-7320-9839
- IdRef : 083944540
Sepideh Valimehr
- Fonction : Auteur
- PersonId : 1346374
- ORCID : 0000-0001-5845-8100
Vincent Chaptal
- Fonction : Auteur
- PersonId : 174577
- IdHAL : vincent-chaptal
- ORCID : 0000-0003-2603-0294
- IdRef : 111506301
Pierre Falson
- Fonction : Auteur
- PersonId : 178243
- IdHAL : pierre-falson
- ORCID : 0000-0002-9760-4577
- IdRef : 03372640X
Marlène Martinho
- Fonction : Auteur
- PersonId : 1223180
- ORCID : 0000-0001-8576-5675
Pierre Dorlet
- Fonction : Auteur
- PersonId : 738342
- IdHAL : pierre-dorlet
- ORCID : 0000-0001-7394-3374
- IdRef : 160857740
Eric Hanssen
- Fonction : Auteur
- PersonId : 1346375
- ORCID : 0000-0002-4064-1844
Jean-Michel Jault
Connectez-vous pour contacter l'auteur
- Fonction : Auteur correspondant
- PersonId : 761296
- ORCID : 0000-0003-1743-2777
- IdRef : 095193324
Connectez-vous pour contacter l'auteur
Cédric Orelle
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- Fonction : Auteur correspondant
- PersonId : 739083
- IdHAL : cedric-orelle
- ORCID : 0000-0003-3418-3290
Connectez-vous pour contacter l'auteur
Résumé
ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.
Domaines
Sciences du Vivant [q-bio]Format du dépôt | Fichier |
---|---|
Type de dépôt | Article dans une revue |
Résumé |
en
ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.
|
Titre |
en
The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains
|
Auteur(s) |
Margot Di Cesare
1
, Elise Kaplan
1
, Julia Rendon
2
, Guillaume Gerbaud
2
, Sepideh Valimehr
3
, Alexia Gobet
1
, Thu-Anh Thi Ngo
1
, Vincent Chaptal
1
, Pierre Falson
1
, Marlène Martinho
2
, Pierre Dorlet
4
, Eric Hanssen
3
, Jean-Michel Jault
1
, Cédric Orelle
1
1
MMSB -
Microbiologie moléculaire et biochimie structurale / Molecular Microbiology and Structural Biochemistry
( 511411 )
- IBCP CNRS/Université de Lyon
7 Passage du vercors
69367 LYON Cedex 07
- France
2
IMM -
Institut de Microbiologie de la Méditerranée
( 186491 )
- 31, chemin Joseph Aiguier
13402 Marseille
- France
3
University of Melbourne
( 306322 )
- Parkville VIC 3010
- Australie
4
BIP -
Bioénergétique et Ingénierie des Protéines
( 457497 )
- CNRS - Université d’ Aix-Marseille
31, chemin Joseph Aiguier
13402 MARSEILLE Cedex 20 FRANCE
Tél : +33 (0)4 91 16 45 50
- France
|
Date de publication électronique |
2023-12-09
|
Vulgarisation |
Non
|
Comité de lecture |
Oui
|
Audience |
Internationale
|
Langue du document |
Anglais
|
Licence |
Paternité
|
Nom de la revue |
|
Date de publication |
2024-01
|
Volume |
300
|
Numéro |
1
|
Page/Identifiant |
105546
|
Projet(s) ANR |
|
Financement |
|
Domaine(s) |
|
Mots-clés |
en
ABC transporter, multidrug resistance, antibiotics, conformational changes, catalytic cycle, transport cycle, drug transport, efflux pump
|
DOI | 10.1016/j.jbc.2023.105546 |
Pubmed Id | 38072053 |
PubMed Central | PMC10821409 |
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