title: Role of a conserved residue R780 in Escherichia coli multidrug transporter AcrB.
abstract: Multidrug efflux pumps play important roles in bacteria drug resistance. A major multidrug efflux system in Gram-negative bacteria is composed of the inner membrane transporter AcrB, outer membrane protein channel TolC, and membrane fusion protein AcrA. These three proteins form a large complex that spans both layers of cell membranes and the periplasmic space. AcrB exists and functions as a homotrimer. To identify residues at the trimer interface that play important roles in AcrB function, we conducted site directed mutagenesis and discovered a key residue, R780. Although R780K was partially functional, all other R780 mutants tested were completely nonfunctional. Replacement of R780 by other residues disrupted trimer association. However, a decrease of trimer stability was not the lone cause for the observed loss of activity, because the activity loss could not be restored by strengthening trimer interaction. Using both heat and chemical denaturation methods, we found that the mutation decreased protein stability. Finally, we identified a repressor mutation, M774K, through random mutagenesis. It restored the activity of AcrBR780A to a level close to that of the wild-type protein. To examine the mechanism of activity restoration, we monitored denaturation of AcrBR780A/M774K and found that the repressor mutation improved protein stability. These results suggest that R780 is critical for AcrB stability. When R780 was replaced by Ala, the protein retained the overall structure, still trimerized in the cell membrane, and interacted with AcrA. However, local structural rearrangement might have occurred and lead to the decrease of protein stability and loss of substrate efflux activity.
authors.affiliation: Department of Chemistry, University of Kentucky ,Lexington, Kentucky 40506, United States.
authors.forename: Linliang
authors.initials: L
authors.lastname: Yu
authors.forename: Wei
authors.initials: W
authors.lastname: Lu
authors.forename: Cui
authors.initials: C
authors.lastname: Ye
authors.forename: Zhaoshuai
authors.initials: Z
authors.lastname: Wang
authors.forename: Meng
authors.initials: M
authors.lastname: Zhong
authors.forename: Qian
authors.initials: Q
authors.lastname: Chai
authors.forename: Michael
authors.initials: M
authors.lastname: Sheetz
authors.forename: Yinan
authors.initials: Y
authors.lastname: Wei
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D055550:Protein Stability; D017433:Protein Structure, Secondary
chemical_list: D000409:Alanine; D029968:Escherichia coli Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; C083496:AcrB protein, E coli