title: Repressive mutations restore function-loss caused by the disruption of trimerization in Escherichia coli multidrug transporter AcrB.
abstract: AcrAB-TolC and their homologs are major multidrug efflux systems in Gram-negative bacteria. The inner membrane component AcrB functions as a trimer. Replacement of Pro223 by Gly in AcrB decreases the trimer stability and drastically reduces the drug efflux activity. The goal of this study is to identify suppressor mutations that restore function to mutant AcrBP223G and explore the mechanism of function recovery. Two methods were used to introduce random mutations into the plasmid of AcrBP223G. Mutants with elevated drug efflux activity were identified, purified, and characterized to examine their expression level, trimer stability, interaction with AcrA, and substrate binding. Nine single-site repressor mutations were identified, including T199M, D256N, A209V, G257V, M662I, Q737L, D788K, P800S, and E810K. Except for M662I, all other mutations located in the docking region of the periplasmic domain. While three mutations, T199M, A209V, and D256N, significantly increased the trimer stability, none of them restored the trimer affinity to the wild type level. M662, the only site of mutation that located in the porter domain, was involved in substrate binding. Our results suggest that the function loss resulted from compromised AcrB trimerization could be restored through various mechanisms involving the compensation of trimer stability and substrate binding.
authors: Department of Chemistry, University of Kentucky Lexington, KY, USA.
authors: Zhaoshuai
authors: Z
authors: Wang
authors: Department of Chemistry, University of Kentucky Lexington, KY, USA.
authors: Meng
authors: M
authors: Zhong
authors: Department of Chemistry, University of Kentucky Lexington, KY, USA.
authors: Wei
authors: W
authors: Lu
authors: Department of Chemistry, University of Kentucky Lexington, KY, USA.
authors: Qian
authors: Q
authors: Chai
authors: Department of Chemistry, University of Kentucky Lexington, KY, USA.
authors: Yinan
authors: Y
authors: Wei
keywords: RND; efflux pump; membrane transporter; oligomerization; protein thermal stability
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