Gram-negative bacteria such as E. coli use tripartite efflux pumps such as
AcrAB-TolC to expel antibiotics and noxious compounds. A key feature of the
inner membrane transporter component, AcrB, is a short stretch of residues known
as the gate/switch loop that divides the proximal and distal substrate binding
pockets. Amino acid substitutions of the gate loop are known to decrease
antibiotic resistance conferred by AcrB. Here we present two new AcrB gate loop
variants, the first stripped of its bulky side chains, and a second in which the
gate loop is removed entirely. By determining the crystal structures of the
variant AcrB proteins in the presence and absence of erythromycin and assessing
their ability to confer erythromycin tolerance, we demonstrate that the gate
loop is important for AcrB export activity but is not required for erythromycin
binding.
