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

Sagar Raturi,Hendrik W Van Veen,Boyan Bai,Keiko Shinoda,Satoshi Murakami,Asha V Nair,Hideaki Fujitani,Himansha Singh

doi:10.1038/s42003-021-02081-6

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.

Highlights

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals
Determination of initial ethidium efflux rates as a function of the Na+ concentration yielded an apparent affinity constant Kt of 0.39 ± 0.06 mM Na+ (Fig. 2c), which is comparable to previously reported data[20]. These results suggest that NorMVC can use both Na+ and H+ as coupling ions. When these experiments were repeated with lactococcal cells expressing the NorM from Vibrio cholerae (NorM-VC) homologue NorM orthologue in Pseudomonas stutzeri (NorM-PS) from P. stutzeri[22,23], the ethidium efflux reaction was stimulated by K+ but not by Na+ (Fig. 2a, b)
Further tests on the transport energetics of the Q278A mutant demonstrate electroneutral ethidium+/H+ antiport (Fig. 9c), and the loss of a proton in the transport reaction compared to wildtype NorM-VC. These results indicate that electrogenic ethidium+/(H+, Na+) antiport and ethidium+/2H+ antiport in NorM-VC require a specific interface between the N-lobe and C-lobe

Summary

Introduction

Multidrug and toxic compound extrusion (MATE) transport proteins confer multidrug resistance on pathogenic microorganisms and affect pharmacokinetics in mammals. MATE transporters are localised in the proximal convoluted tubule and proximal straight tubule in the kidney, as well as the canalicular membrane in hepatocytes, where they mediate transport of organic cations in the final steps of drug elimination from the body[3,5]. The ion/drug stoichiometries and dependencies of the antiport reactions on chemical ion gradients and the membrane potential are mostly unknown Using these methods in our previous study, we identified the ability of NorM from Vibrio cholerae (NorM-VC) to utilise both the H+ and Na+ gradient in its drug-efflux activity[20]. We prepared NorM-VC/NorM-PS chimaeras and mutant proteins that enabled us to dissect the localisation of ion-coupling events and roles of conserved catalytic carboxylates in H+ binding and Na+ coordination

Methods

Results

Conclusion