β-Lactam Selectivity of Multidrug Transporters AcrB and AcrD Resides in the Proximal Binding Pocket

Background: Bacterial multidrug transporters AcrB and AcrD export -lactam antibiotics. Results: Charged residues in the proximal binding pocket play a crucial role in -lactam selectivity. Conclusion: Proximal pocket acts as a substrate selection filter at the site of entry into the substrate translocation pathway. Significance: Our understanding of molecular mechanisms of antibiotic recognition is essential for overcoming drug resistance. -Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic -lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in -lactam specificity between AcrB and AcrD. We identified three crucial residues at the proximal (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the -lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in -lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.