Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB.
The AcrA-AcrB-TolC complex is the major multidrug efflux pump in Escherichia coli. The asymmetric structure of the trimeric inner-membrane component AcrB implies functional rotation of the monomers and a peristaltic mode of drug efflux. This mechanism suggests the occurrence of conformational changes in the periplasmic pore domain through the movements of subdomains during cycling of the monomers through the different states loose (L), tight (T) and open (O). We introduced cysteines at the interfaces of potentially moving subdomains, leading to disulfide bond formation as quantified by alkylation of free cysteines and MALDI-TOF analysis. Inhibition of pump function as a result of cross-linking caused increased susceptibility to noxious compounds and reduction of N-phenylnaphthylamine efflux. Regain of function for impaired mutants was obtained upon exposure to the reducing agent DTT. The results support the presence of the asymmetric AcrB trimer in E. coli membranes and the functional rotation mechanism.
Seeger
Markus A
MA
Institute of Physiology and Zurich Centre for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
von Ballmoos
Christoph
C
Eicher
Thomas
T
Brandstätter
Lorenz
L
Verrey
François
F
Diederichs
Kay
K
Pos
Klaas M
KM
D015057:1-Naphthylamine / Q000031:analogs & derivatives / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D003545:Cysteine / Q000235:genetics; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism*; D017353:Gene Deletion; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019163:Reducing Agents / Q000494:pharmacology
D015057:1-Naphthylamine; D000900:Anti-Bacterial Agents; D003545:Cysteine; D004220:Disulfides; D004229:Dithiothreitol; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D050505:Mutant Proteins; D019163:Reducing Agents; C083496:AcrB protein, E coli; C005444:N-phenyl-1-naphthylamine