Dissection of Protonation Sites for Antibacterial Recognition and Transport in QacA, a Multi-Drug Efflux Transporter.

QacA is a drug:H+ antiporter with 14 transmembrane helices that confers antibacterial resistance to methicillin-resistant Staphylococcus aureus strains, with homologs in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+-driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in the identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis-based assays. The activity of purified QacA was also observed using proteoliposome-based substrate-induced H+ transport assay. Our results identify two sites, D34 and D411 as vital players in substrate recognition, while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays an additional role as a substrate recognition site for the transport of dequalinium, a divalent quaternary ammonium compound. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.