A Three-Dimensional Model for the Substrate Binding Domain of the Multidrug ATP Binding Cassette Transporter LmrA

GF Ecker, K Pleban, S Kopp, E Csaszar, GJ Poelarends, M Putman, D Kaiser, WN Konings, P Chiba*

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

25 Citations (Scopus)

Abstract

Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the alpha-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.

Original languageEnglish
Pages (from-to)1169 - 1179
Number of pages11
JournalMolecular Pharmacology
Volume66
Issue number5
DOIs
Publication statusPublished - Nov-2004

Keywords

  • RESISTANCE P-GLYCOPROTEIN
  • LACTOCOCCUS-LACTIS
  • DRUG-BINDING
  • TRANSMEMBRANE DOMAINS
  • MASS-SPECTROMETRY
  • ESCHERICHIA-COLI
  • ABC TRANSPORTER
  • ANTIBIOTIC-RESISTANCE
  • NUCLEOTIDE-BINDING
  • TRANSITION-STATE

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