Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter.

QacA is a drug:H+ antiporter (DHA2) with 14 transmembrane helices, that renders antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) strains, with homologues 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 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. We 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 a moonlighting role as a substrate recognition site for dequalinium transport. 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.