Covalently Linked Trimers of RND (Resistance-Nodulation-Division) Efflux Transporters to Study Their Mechanism of Action: Escherichia coli AcrB Multidrug Exporter as an Example
Transporters undergo large conformational changes in their functional cycle. RND (Resistance-Nodulation-Division) family efflux transporters usually exist as homotrimers, and each protomer was proposed to undergo a cycle of conformational changes in succession so that at any given time the trimer would contain three protomers of different conformations, the functionally rotating mechanism of transport. This mechanism implies that the inactivation of one protomer among three will inactivate the entire trimeric ensemble by blocking the functional rotation. We describe a biochemical approach to test this prediction by first producing a giant protein in which the three protomers of Escherichia coli AcrB efflux pump are covalently linked together through linker sequences, and then testing for its function by inactivation of a single protomer unit. Inactivation can be done permanently by mutating a residue involved in proton relay, or in “real time” by using a protein in which one protomer contains two Cys residues on both sides of the large cleft in the periplasmic domain and then by rapidly inactivating this protomer with a methanethiosulfonate cross-linker.
Key wordsRND family transporters Antibiotics Drug efflux Disulfide bonds Methanethiosulfonate AcrB Proton relay Functionally rotating mechanism
This work was supported by a grant from the U.S. Public Health Service (AI-009644). I thank Y. Takatsuka for supplying the details and for suggestions.
- 5.Sennhauser G, Amstutz P, Briand C et al (2007) Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol e7:5Google Scholar
- 10.Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar