Engineered MATE multidrug transporters reveal two functionally distinct ion-coupling pathways in NorM from Vibrio cholerae

Abstract

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. Our understanding of how MATE transporters work, has mostly relied on protein structures and MD simulations. However, the energetics of drug transport has not been studied in detail. Many MATE transporters utilise the electrochemical H+ or Na+ gradient to drive substrate efflux, but NorM-VC from Vibrio cholerae can utilise both forms of metabolic energy. To dissect the localisation and organisation of H+ and Na+ translocation pathways in NorM-VC we engineered chimaeric proteins in which the N-lobe of H+-coupled NorM-PS from Pseudomonas stutzeri is fused to the C-lobe of NorM-VC, and vice versa. Our findings in drug binding and transport experiments with chimaeric, mutant and wildtype transporters highlight the versatile nature of energy coupling in NorM-VC, which enables adaptation to fluctuating salinity levels in the natural habitat of V. cholerae.

Energetics of lipid transport by the ABC transporter MsbA is lipid dependent

“…In addition to lipids, MsbA transports small molecules like ethidium, erythromycin and chloramphenicol 10 , 11 , 15 . The efflux of ethidium can conveniently be monitored in L. lactis cells from the decrease in fluorescence emission when the dye dissociates from intracellular nucleic acids 27 . In contrast to the observations for Lipid-A and PE, the R78A R148A double mutant and TripRA mutant showed a significantly enhanced efflux of ethidium compared to MsbA-WT (Fig.…”

CHARMM-GUI Membrane Builder: Past, Current, and Future Developments and Applications

“…As some transporters are driven by proton and/or Na + gradients, it becomes important to identify the proton and Na + binding sites. Raturi et al studied the binding and disassociation of Na + in a MATE (multidrug and toxic compound extrusion) transporter by simulating the transporter with Na + prebound and by letting the Na + freely diffuse into the sites . Zhao et al similarly studied the Na + binding in another MATE transporter .…”

“…Raturi et al studied the binding and disassociation of Na + in a MATE (multidrug and toxic compound extrusion) transporter by simulating the transporter with Na + prebound and by letting the Na + freely diffuse into the sites. 203 Zhao et al similarly studied the Na + binding in another MATE transporter. 204 Bavnho̷ j et al, on the other hand, used constant pH simulations to determine the protonation states of sugar transporter protein STP10 and established the basis for proton-to-glucose coupling.…”

The culmination of multidrug-resistant efflux pumps vs. meager antibiotic arsenal era: Urgent need for an improved new generation of EPIs

“…For a variety of membrane transporters, alternating access mechanisms have shown evolutionary persistence over the past few decades (Jardetzky, 1966 ). During sequential conformational modifications, a substrate-binding pocket in the middle of the transporter is exposed to the inner cellular space, while the other exposes the pocket to the outer cellular space through multiple conformational changes (Raturi et al, 2021 ). However, the structure and catalysis of MATE transporters underlying conformational changes are still not clearly understood.…”

“…However, the structure and catalysis of MATE transporters underlying conformational changes are still not clearly understood. Partly, this is due to the fact that, particularly, two previously reported crystal structures of MATE transporters were with an outward-facing orientation (Raturi et al, 2021 ). Therefore, the MATE transporters facilitating drug/ion coupled transportation via Na+ or H+ coupling (Lu et al, 2013a ) have already been described.…”