Antibiotic export by MexB multidrug efflux transporter is allosterically controlled by a MexA-OprM chaperone-like complex

Abstract

The tripartite multidrug efflux system MexAB-OprM is a major actor in Pseudomonas aeruginosa antibiotic resistance by exporting a large variety of antimicrobial compounds. Crystal structures of MexB and of its Escherichia coli homolog AcrB had revealed asymmetric trimers depicting a directional drug pathway by a conformational interconversion (from Loose and Tight binding pockets to Open gate (LTO) for drug exit). It remains unclear how MexB acquires its LTO form. Here by performing functional and cryo-EM structural investigations of MexB at various stages of the assembly process, we unveil that MexB inserted in lipid membrane is not set for active transport because it displays an inactive LTC form with a Closed exit gate. In the tripartite complex, OprM and MexA form a corset-like platform that converts MexB into the active form. Our findings shed new light on the resistance nodulation cell division (RND) cognate partners which act as allosteric factors eliciting the functional drug extrusion.

MexAB-OprM Efflux Pump Interaction with the Peptidoglycan of Escherichia coli and Pseudomonas aeruginosa

“…We recently solved the structure of the whole MexAB-OprM pump from P. aeruginosa by cryo-EM [ 16 ] showing a structure of around 230 Å long between the two membranous domains, which is compatible with the size of the periplasm estimated by cryo-transmission electron microscopy (Cryo-TEM) [ 36 ]. Matias et al measured frozen-hydrated sections of E. coli and P. aeruginosa showing some differences between the two bacteria.…”

“…The constitutive RND efflux pumps MexAB-OprM from P. aeruginosa and AcrAB-TolC from Escherichia coli (E. coli) have been extensively studied by different approaches making them archetypal models for the structural and functional comprehension of the efflux pump mechanism. The structure of each protein forming the pump has been solved by X-ray crystallography [4][5][6][7][8][9][10][11][12] and the whole assembly was recently determined by cryo-electron microscopy (cryo-EM) [13][14][15][16].…”

“…The constitutive RND efflux pumps MexAB-OprM from P. aeruginosa and AcrAB-TolC from Escherichia coli ( E. coli) have been extensively studied by different approaches making them archetypal models for the structural and functional comprehension of the efflux pump mechanism. The structure of each protein forming the pump has been solved by X-ray crystallography [ 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 ] and the whole assembly was recently determined by cryo-electron microscopy (cryo-EM) [ 13 , 14 , 15 , 16 ]. The comparison of these structures from several homologous pumps, together with in vitro measurements of transport through proteoliposomes [ 17 , 18 ], in cellulo determination of the minimal inhibitory concentration (MIC) of antibiotics for mutant strains [ 19 , 20 ], and the extensive modeling and molecular dynamic calculations [ 21 , 22 ], have led to a more precise understanding of the efflux pathways [ 23 , 24 , 25 , 26 , 27 , 28 ] and the conformational modifications [ 14 , 29 , 30 ] necessary to expulse the drugs out of the cell.…”

“…We know now that the RND protein works as homotrimer with a proton motive force via proton relay amino acids localized in the membranous domain. This proton transfer is combined with the movement of the RND periplasmic domains where each monomer adopts alternatively Loose, Tight, and Close or Open conformations [ 31 ], to capture the drug at different entries and push it toward the RND funnel domain [ 16 , 32 ]. The drug then crosses the periplasm by a tunnel formed by the hexameric MFP proteins and is finally expulsed out of the cell through the OMF channel.…”

Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy

“…Most of the structures have been obtained by SP cryo-EM analysis (316 unique MP structures, 587 reports). The number of α -helical MPs almost doubles, with 536 reports versus 281 unique structures (six reports missing from S. White's database have been included in the list (Wang and Sigworth, 2009; Glavier et al ., 2020; Hua et al ., 2020; McDowell et al ., 2020; Yao et al ., 2020; Zhang et al ., 2020)). Here again, the most representative subgroups include channels, ABC transporters, electron transport chain supercomplexes, ATPases, and GPCRs.…”