Dissecting the molecular mechanism of an antibiotic efflux pump – BIOTIFLUX

cryo-electron microscopy, X-ray crystallography, ATPase and transport assays, Hydrogen/Deuterium exchange coupled to mass spectrometry, EPR spectroscopy and solid-state NMR

We have solved the X-ray and cryoEM structures of BmrA in complex with ATP-Mg2+ and rhodamine 6G (submitted). We have reconstituted BmrA in nanodiscs and studied its conformational dynamics by Hydrogen/Deuterium exchange coupled to mass spectrometry (HDX). We could thus observe the conformational changes in different steps of the catalytic cycle: apo (no ligand), transition state (ADP-Mg2+-Vi), and drug-bound (doxorubicin). These results allowed us to identify (i) flexible regions of the transmembrane domains in the outward-facing conformation (ii) important amino acids involved in the communication between the nucleotide-binding domains (NBDs) and the transmembrane domains (TMDs) (iii) regions of the protein that respond to doxorubicin binding. Finally, we investigated how different ATP Analogues can be used for structural investigations of BmrA.

We are continuing to acquire structural, biochemical and biophysical data on several key steps of the transport mechanism of BmrA. In-fine, these data will give us insights on the allosteric transition ongoing during ATP-binding, and the transitions from the inward to the outward-facing conformations.

1. Structures of ABC transporters: handle with care. Oded Lewinson, Cédric Orelle and Markus A. Seeger. FEBS let. (2020), 594(23):3799-3814.

2. ATP Analogues for Structural Investigations: Case Studies of a DnaB Helicase and an ABC Transporter. Denis Lacabanne, Thomas Wiegand, Nino Wili, Maria I Kozlova, Riccardo Cadalbert, Daniel Klose, Armen Y Mulkidjania, Beat H Meier and Anja Böckmann. Molecules (2020);25(22):5268.

3. Identification of novel inhibitors of the ABC transporter BmrA. Onur Serçinoglu, Duygu Senturk, Fatma E. Altinisik Kaya, Fatma Gizem Avci, Rok Frlan, Tihomir Tomašic, Pemra Ozbek, Cédric Orelle, Jean-Michel Jault and Berna Sariyar Akbulut. Bioorg Chem (2020);105:104452.

4. Functional overexpression of membrane proteins in E. coli: the good, the bad and the ugly. Margot Di Cesare, Aissatou Maty Diagne, Benjamin Bourgey, Jean-Michel Jault and Cédric Orelle. Methods Mol. Biol. (2021); in press.

Antibiotic resistance is becoming a major threat for human health. It represents a difficult challenge to address, especially with the outcome of multidrug resistance (MDR), when bacteria become resistant to multiple structurally and functionally unrelated molecules. Drug efflux pumps contribute to this phenomenon and can promote bacterial adaptation by favoring the emergence of other resistance mechanisms. An increasing number of bacterial ABC transporters, which utilize the energy of ATP hydrolysis to expel drugs, have been implicated in multidrug resistance mechanisms. Because their functioning mechanism remains unclear, we propose to decrypt the molecular mechanism of one of this pump, BmrA from Bacillus subtilis, by using a combination of structural and conformational dynamics approaches.