Insights into substrate recognition and export tunnel preferences in the efflux transporter AcrB

In Escherichia coli AcrB is a major multidrug exporter, which confers the bacterium resistance to many antibiotics with diverse structural and chemical proprieties. Studies have identified three possible tunnels (or channels) within AcrB that different substrates use before reaching the distal pocket, from which they are subsequently extruded. Recently, we reported that mutations in the AcrB gate loop may affect the conformational change kinetics involved in substrate export rather than directly affecting molecular interactions with this loop, and we highlighted the distinct export tunnel preferences between erythromycin and doxorubicin. To further understand the gate loop's role in AcrB's export activity and the rationale behind substrate preferences among the three possible export tunnels, namely tunnel-1, -2, and -3, we investigated the structural and functional effects of several single and multiple mutations in the gate loop of AcrB. Our findings indicate that all three tunnels are energetically favorable for the substrates studied, with the majority forming more hydrogen bonds in any tunnel compared to the distal pocket. Moreover, our experimental and computational data revealed that some substrates with high molecular similarity exhibited different export tunnel preferences, as strongly suggested by their MIC values. To explain this unexpected outcome, we propose a generalized explanation that the conformational change kinetics in AcrB is substrate-dependent.