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On the mechanisms of transport and energy coupling in ABC exporters 10.17863/CAM.23391 Singh, Himansha van Veen , Dr. Hendrik The rapid emergence of multidrug resistant bacterial strains represents a major global healthcare issue. Amongst five known classes of membrane transporters, which play a huge role in multidrug efflux, primary-active ATP-binding cassette (ABC) transporters are ATP powered whilst secondary-active transporters utilize electrochemical ion gradients to drive substrate transport. Mechanistic insights into transport by these proteins can help with the design and development of novel therapeutic agents against multidrug resistance, and can increase our understanding of the physiological functions of these transporters. Although available crystal structures illustrate a common alternate access model for transport by ABC transporters, the mechanisms by which metabolic energy is coupled to the transport cycle is still elusive. This thesis presents a series of functional studies using whole cells as well as artificial phospholipid membranes to study the energetics of transport, and the influence of membrane phospholipids on substrate transport by the homodimeric Escherichia coli lipid A/multidrug ABC exporter MsbA. Current alternating access models for ABC exporters involve cycling between conformations with inward- and outward-facing substrate-binding sites in membrane domains (MDs) in response to engagement and hydrolysis of ATP at the nucleotide-binding domains (NBDs). Here we report that MsbA also utilizes another major energy currency in the cell by coupling substrate transport to a transmembrane electrochemical proton gradient. In this thesis, analogous substrate transport reactions are also studied for two other ABC exporters, the MsbA homologue LmrA and the human multidrug transporter ABCG2. The dependence of ATP-dependent transport on proton coupling, and the stimulation of MsbA-ATPase by the chemical proton gradient highlight the functional integration of both forms of metabolic energy. It also raises questions about the role of NBDs in the transport process. Comparisons of drug transport and resistance in cells expressing MsbA-MD (truncated MsbA lacking the NBD) and full length MsbA (MsbA-WT) demonstrate increased transport efficiency of MsbA-WT compared to MsbA-MD. In addition, growth studies using E. coli WD2 cells, which are conditionally defective in MsbA’s essential activity in lipid A transport, show that lipid A transport can be restored by the expression of MsbA-WT but not MsbA-MD or ATP-hydrolysis impaired Walker A mutant (MsbA- ΔK382). Lastly, we also present biochemical experiments with proteoliposomes with a defined phospholipid composition, which suggest that cardiolipin is essential for the transport activity of MsbA. These techniques open the way to further explore lipid-proteins interactions and examine the physiological role(s) of MsbA. In conclusion, this thesis produces new insights in the mechanisms of transport and energy coupling in ABC exporters University of Cambridge 2018-07-21 Thesis Doctoral Doctor of Philosophy (PhD) en 1) Cambridge International Scholarship Scheme 2) Rajiv Gandhi Foundation https://www.repository.cam.ac.uk/handle/1810/276108 controlled.access https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a7585c9d-e26b-466b-a865-279b421274f6/download 87eda9de84448d1f82354d60eee3eb5f https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/a556d7d2-5d56-4a61-bc2a-883600ae45e7/download 83df02a2c0483d319dba449f0be4022c https://www.rioxx.net/licenses/all-rights-reserved/ ABC transporters Bioenergetics ion transport membrane proteins Antibiotic resistance