Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB - PubMed (original) (raw)

Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB

Jürgen A Bohnert et al. J Bacteriol. 2008 Dec.

Abstract

The Escherichia coli multidrug efflux pump protein AcrB has recently been cocrystallized with various substrates, suggesting that there is a phenylalanine-rich binding site around F178 and F615. We found that F610A was the point mutation that had the most significant impact on substrate MICs, while other targeted mutations, including conversion of phenylalanines 136, 178, 615, 617, and 628 to alanine, had smaller and more variable effects.

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Figures

FIG. 1.

Western blot analysis of mutant AcrB production. Total protein extracts of E. coli 3-AG100 mutants (14 μg protein) were separated by NuPAGE Novex bis-Tris (Invitrogen, California) gel electrophoresis and probed with polyclonal anti-AcrB antibodies. Lanes MW contained molecular weight markers.

FIG. 2.

Increases in EtBr (a) and PAβN (b) fluorescence in AcrB phenylalanine mutants compared to pseudomutant AcrB strain F628F. Fluorescence was recorded for 30 min after addition of 2.5 μM EtBr or 200 μM PAβN. The values are means of at least duplicate experiments. RFU, relative fluorescence units.

FIG. 3.

AcrB binding pocket based on the “tight” monomer 2GIF structure coordinates (12). Phenylalanines are indicated by sticks. The image was generated using the molecular visualization software PyMol (

http://pymol.sourceforge.net

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Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB - PubMed (original) (raw)