title: 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.
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: Martha
authors.initials: M
authors.lastname: Radchenko
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064.
authors.forename: Rongxin
authors.initials: R
authors.lastname: Nie
authors.affiliation: From the Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois 60064 min.lu@rosalindfranklin.edu.
authors.forename: Min
authors.initials: M
authors.lastname: Lu
mesh_terms: D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001693:Biological Transport, Active; D004220:Disulfides / Q000737:chemistry*; D009344:Neisseria gonorrhoeae / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary
chemical_list: D017920:Antiporters; D001426:Bacterial Proteins; D004220:Disulfides; C114619:NorM protein, bacteria
keywords: conformational change; cysteine cross-linking; drug resistance; exchanger; membrane protein; multidrug efflux; multidrug transporter