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

Publication date: Available online 23 March 2019Source: Journal of Molecular BiologyAuthor(s): Puja Majumder, Shashank Khare, Arunabh Athreya, Nazia Hussain, Ashutosh Gulati, Aravind PenmatsaAbstractQacA is a drug:H+ antiporter (DHA) with 14 transmembrane helices, that confers antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) 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 quate...
Source: Journal of Molecular Biology - Category: Molecular Biology Source Type: research