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.
Highlights
Multidrug and toxic compound extrusion (MATE)[3] proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar, typically cationic
Our experiments demonstrated that copper phenanthroline abrogated the ability of the five cross-linkable mutants to confer drug resistance, likely by promoting the formation of the disulfide bonds (Fig. 6B), which inactivated NorM from Neiserria gonorrheae (NorM-NG) by precluding any further structural changes required for multidrug efflux
These amino acid substitutions exerted little effect on the expression or transport function of NorM-NG (Figs. 2, 6, and 7), we found copper phenanthroline substantially altered the migration of the purified dicysteine mutants in SDS-PAGE (Fig. 3A)
Summary
Multidrug and toxic compound extrusion (MATE)[3] proteins constitute a ubiquitous family of multidrug transporters and couple the efflux of structurally dissimilar, typically cationic. Over the past 6 years, the x-ray structures of Naϩ-coupled NorM from Vibrio cholera and Neiserria gonorrheae (NorM-VC and NorM-NG), and Hϩ-coupled DinF from Pyrococcus furiosus and Bacillus halodurans (PfMATE and DinFBH) have been reported, revealing the transporter architecture and providing important insights into the underlying transport mechanisms (6 –10). These findings highlight new areas of uncertainty that need to be addressed experimentally (11). Our findings take our understanding of the MATE-mediated multidrug efflux to new depths and lend fresh hope for thwarting multidrug resistance
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