Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux

Abstract

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism.

Multidrug efflux pumps: structure, function and regulation

“…The structures apparently adopt an outward-open state with the lower portion of the central cavity situated about halfway through the membrane bilayer. NorM may use an alternative access transport mechanism, as indicated by the proximity of residue pairs in vivo 59 .…”

The N-terminal domain of an archaeal multidrug and toxin extrusion (MATE) transporter mediates proton coupling required for prokaryotic drug resistance

“…Tetraphenylphosphonium, ethidium, and rhodamine 6G (R6G) were found to bind a negatively charged central cavity formed by acidic and polar residues from the NTD and CTD near the membrane-water interface in the Na ϩ -coupled transporter NorM from Neisseria gonorrhoeae (20). Ligand-dependent conformational changes were predicted to facilitate an allosteric coupling transport mechanism (20,29). In contrast, R6G was found buried within the hydrophobic TM core of H ϩ -coupled DinF from Bacillus halodurans (DinF-BH), in which the majority of contacts are made with NTD residues, including a conserved Asp in TM1 that mediates the only charge-charge interaction with the substrate (19).…”

Functional insights of two MATE transporters fromVibrio fluvialis

“…Subsequently, molecular docking was done using SwissDock (http://www.swissdock.ch/) to validate binding interactions of both the transporters with ethidium bromide and norfloxacin as both the transporters showed certain degree of transport for these substrates. Classically, a MATE pump has 12 TMs arranged in two bundles of six TMs each (Tanaka et al, 2013, Tanaka et al, 2017, Radchenko et al, 2016, Lu et al, 2013b. The first TM has an ion binding site represented by D32, the Aspartic Acid residue at 32 position (Otsuka et al, 2005, Long et al, 2008, Lu et al, 2013a, Lu et al, 2013b.…”