Structural and Functional Investigation of a Multi-drug Efflux Transporter QacA
The emergence of multi-drug resistance in bacteria is a global health care challenge. One of the effective means of gaining antimicrobial resistance, among superbugs, is through expression of efflux pumps. Quaternary ammonium compound transporters, QacA/B, that are observed in Staphylococcus aureus strains are capable of transporting 30 chemically dissimilar monovalent and divalent cationic antibacterial compounds and dyes. The 14-TM (transmembrane) helix containing transporter QacA, belongs to the drug/H+ antiporter 2 (DHA2) family which is a part of major facilitator superfamily (MFS). MFS transporters are the largest superfamily of secondary active transporters. QacA utilizes the H+ gradient across the bacterial cell membrane for the uphill efflux of the cytotoxic compounds. QacA has two distinct TM domains, each of which consists of 6-TM helices that retain a pseudo 2-fold symmetry amongst them. During the transport process, the domains move in rocker-switch mechanism to allow alternating-access to either side of the membrane in order to transport the substrates. The expression of this efflux pump is regulated by a trans-acting regulatory DNA binding protein QacR. Under normal condition, QacR blocks the transcription of qacA gene by binding to the operator DNA but under antibacterial stress, the substrates of QacA binds to QacR, causing dissociation of QacR from the operator DNA and QacA gets expressed. In this thesis, structural and functional investigation of QacA was carried out and some fundamental questions about this multi-drug efflux transporter are addressed. In the first part, wild-type QacA was purified and the transport activity of the transporter both in native membrane and in isolation was analyzed using substrate-induced H+-release assay and a reconstitution-based assay. The binding studies with the cytotoxic substrates (TPP, Pm, Dq) displayed sub-millimolar binding affinity with the purified transporter and substrate/H+ competition assay suggested the presence of substrate-protonation site interactions in QacA. Further, survival assays done in the presence of TPP and Pm and whole cell ethidium efflux assay illustrated that ΔpH provides primary driving energy to the transporter. In the second part of the thesis, six protonatable acidic residues D34, D61, D323, E406, E407 and D411, lining the transport vestibule were identified using a homology model of QacA and each of the residue was characterized using mutagenesis. The binding studies and the transport assays illustrated D34, D323, E407 and D411 are crucial for the transport activity of the transporter either as substrate recognition sites or indirectly facilitating the transport process as protonation sites. The findings of the study suggested the inherent residue level promiscuity for different substrates of QacA, that can explain broad substrate specificities of QacA and other related multi-drug efflux transporters. The third part of the thesis described single-domain Indian camelid antibody (ICab) library generation and isolation of high affinity binders against QacA, in order to stabilize the transporter to facilitate the structural studies. The sorting of the binding population was done using yeast surface display coupled with flow-cytometry and 7 unique ICab binders were isolated. The last part of the thesis is focused on the heterologous expression and purification of two of the camelid antibodies in E. coli through refolding and cytosolic protein preparation. The binding studies using FSEC (fluorescence detection size exclusion chromatography), flow-cytometry and microscale thermophoresis suggested that the purified ICabs bind to the transporter with nanomolar affinity. Furthermore, 2D classes from cryo-electron microscopy of QacA-ICab complex clearly displayed the presence of the transporter in detergent micelles bound to the ICab. Moreover, the effect of ICab on QacA-substrate interactions indicated that ICabs can block substrate binding to the transporter. The results provide an interesting prospect of using the ICabs as efflux pump inhibitors (EPI).