A three-dimensional model for the substrate binding domain of the multidrug ATP binding cassette transporter LmrA.
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.
Ecker
Gerhard F
GF
Institute of Medical Chemistry, Medical University of Vienna, Waehringerstrasse 10, A-1090 Vienna, Austria.
Pleban
Karin
K
Kopp
Stephan
S
Csaszar
Edina
E
Poelarends
Gerrit J
GJ
Putman
Monique
M
Kaiser
Dominik
D
Konings
Wil N
WN
Chiba
Peter
P
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001692:Biological Transport; D013294:Lactococcus lactis / Q000378:metabolism*; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels; D011405:Propafenone / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D019920:Photoaffinity Labels; D011405:Propafenone; C101824:LmrA protein, Lactococcus lactis