PT - JOURNAL ARTICLE AU - Puja Majumder AU - Shashank Khare AU - Arunabh Athreya AU - Nazia Hussain AU - Ashutosh Gulati AU - Aravind Penmatsa TI - Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter AID - 10.1101/443523 DP - 2018 Jan 01 TA - bioRxiv PG - 443523 4099 - http://biorxiv.org/content/early/2018/10/15/443523.short 4100 - http://biorxiv.org/content/early/2018/10/15/443523.full AB - 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.HighlightsA homology model of QacA with 14 TM helices was used to test the importance of acidic residues within the vestibule.Mono and Divalent cationic substrate recognition requires two sites D34 (TM1) and D411 (TM13).E407 (TM13) is important for protonation driven efflux.Substrate recognition of a divalent substrate, dequalinium occurs at E407 instead of D411 providing glimpses into the promiscuity of substrate recognition in QacA.AbbreviationsACMA9-Amino-6-Chloro-2-MethoxyacridineTMTransmembraneDHADrug-Proton Antiporter/Drug-H+ AntiporterEtEthidiumTPPTetraphenylphosphoniumDqDequaliniumPmPentamidineCxChlorhexidineCCCPCarbonyl cyanide m-chlorophenyl hydrazonePiPropamidinePOTProton-dependent Oligopeptide TransporterλEx/λEmExcitation/Emission WavelengthMSTMicroscale ThermophoresisELExtracellular LoopFSECFluorescence-detection Size Exclusion ChromatographyUDMUndecyl-β-D-MaltopyranosidePEG-MalPolyethylene Glycol–MaleimideMALSMulti-Angle Light Scattering.