{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/369954"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/369954","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Investigations into the Lipid Transport Mechanisms and Energetics of Bacterial ATP-Binding Cassette (ABC) Transporters MsbA and LmrA","abstract":"LmrA and MsbA are two paradigms in research on multidrug ATP-binding cassette (ABC) transporters. The bacterial transmembrane protein LmrA from *Lactococcus lactis* was the first known prokaryotic ABC transporter homologue of the mammalian multidrug resistance transporter ABCB1 (P-glycoprotein) characterised to have multidrug transport activity. A distinctive feature of LmrA as a primary-active transporter is its ability to harness electrochemical ion gradients. When measured in an electrophysiological setting, LmrA exhibits ion conductance upon the binding of ATP, which is based on a 2Na<sup>+</sup>/(1H<sup>+</sup>-1HEPES<sup>+</sup>-1Cl−)<sup>+</sup> exchange reaction. It also shows a Na<sup>+</sup>/ethidium<sup>+</sup> antiport reaction that can be driven by the sodium-motive force, which when directed inwardly in cells gives rise to enhanced ethidium efflux, and when directed outwardly in proteoliposomes gives rise to ethidium uptake by purified inside-out oriented LmrA. However, a detailed understanding of the sodium coupling mechanisms of LmrA is limited. LmrA has also been shown to mediate the transport of phospholipids, but the energetics of this process is unknown. To further examine the role of the sodium-motive force in the transport of a broader range of substrates, this PhD study investigated the transport of long-acyl-chain (2 x C18) headgroup biotin-labelled phosphatidylethanolamine (PE) by LmrA. In addition, the potential involvement of polar and acidic residues within LmrA in interaction with Na<sup>+</sup> was assessed. MsbA mediates the translocation of core lipopolysaccharides (core-LPS), hexa-acylated Lipid-A modified with the core oligosaccharides, as well as phospholipids across the plasma membrane in Gram-negative bacteria. It is essential for cell envelope integrity and has become an attractive target for the development of novel antibiotics against pathogenic bacteria. MsbA can also function as a multidrug transporter, sharing a broad substrate specificity for various drugs and cytotoxic agents with LmrA and ABCB1. Previously, using proteoliposomes containing purified MsbA and custom-made biotinylated-Lipid-A, the ability of MsbA to transport Lipid-A in an ATP-dependent fashion was biochemically demonstrated for the first time. Similar to the transport of small-molecule drugs, the flopping of physiologically relevant long-acyl-chain (2 x C18) headgroup biotin-labelled PE in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and a chemical proton gradient as sources of metabolic energy. The energetics of Lipid-A and PE transport was further investigated in this PhD study. Furthermore, the function of the recently reported peripheral (Kdo)2-Lipid-A (KDL) binding sites in MsbA near the cytoplasmic leaflet of the plasma membrane was explored. Unlike the binding of core-LPS in the central cavity of MsbA, which has been identified as an intermediate state in the translocation cycle, the relevance of the core-LPS binding at the periphery of MsbA to the lipid translocation process is less clear. This PhD study utilised a wide range of methods, including cell biology, mutational analyses, biochemical assays, and molecular dynamics simulations to investigate the transport mechanisms of LmrA and MsbA. The findings enhance our understanding of the transport activities of bacterial multidrug ABC transporters, which may ultimately offer insights into the development of novel antibiotics that bypass or inhibit bacterial multidrug efflux pumps. As LmrA and MsbA are bacterial homologues of ABCB1, these insights could also contribute to the mechanistic studies of mammalian multidrug ABC transporters.","abstract_html":"LmrA and MsbA are two paradigms in research on multidrug ATP-binding cassette (ABC) transporters. The bacterial transmembrane protein LmrA from *Lactococcus lactis* was the first known prokaryotic ABC transporter homologue of the mammalian multidrug resistance transporter ABCB1 (P-glycoprotein) characterised to have multidrug transport activity. A distinctive feature of LmrA as a primary-active transporter is its ability to harness electrochemical ion gradients. When measured in an electrophysiological setting, LmrA exhibits ion conductance upon the binding of ATP, which is based on a 2Na&lt;sup&gt;+&lt;/sup&gt;/(1H&lt;sup&gt;+&lt;/sup&gt;-1HEPES&lt;sup&gt;+&lt;/sup&gt;-1Cl−)&lt;sup&gt;+&lt;/sup&gt; exchange reaction. It also shows a Na&lt;sup&gt;+&lt;/sup&gt;/ethidium&lt;sup&gt;+&lt;/sup&gt; antiport reaction that can be driven by the sodium-motive force, which when directed inwardly in cells gives rise to enhanced ethidium efflux, and when directed outwardly in proteoliposomes gives rise to ethidium uptake by purified inside-out oriented LmrA. However, a detailed understanding of the sodium coupling mechanisms of LmrA is limited. LmrA has also been shown to mediate the transport of phospholipids, but the energetics of this process is unknown. To further examine the role of the sodium-motive force in the transport of a broader range of substrates, this PhD study investigated the transport of long-acyl-chain (2 x C18) headgroup biotin-labelled phosphatidylethanolamine (PE) by LmrA. In addition, the potential involvement of polar and acidic residues within LmrA in interaction with Na&lt;sup&gt;+&lt;/sup&gt; was assessed. MsbA mediates the translocation of core lipopolysaccharides (core-LPS), hexa-acylated Lipid-A modified with the core oligosaccharides, as well as phospholipids across the plasma membrane in Gram-negative bacteria. It is essential for cell envelope integrity and has become an attractive target for the development of novel antibiotics against pathogenic bacteria. MsbA can also function as a multidrug transporter, sharing a broad substrate specificity for various drugs and cytotoxic agents with LmrA and ABCB1. Previously, using proteoliposomes containing purified MsbA and custom-made biotinylated-Lipid-A, the ability of MsbA to transport Lipid-A in an ATP-dependent fashion was biochemically demonstrated for the first time. Similar to the transport of small-molecule drugs, the flopping of physiologically relevant long-acyl-chain (2 x C18) headgroup biotin-labelled PE in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and a chemical proton gradient as sources of metabolic energy. The energetics of Lipid-A and PE transport was further investigated in this PhD study. Furthermore, the function of the recently reported peripheral (Kdo)2-Lipid-A (KDL) binding sites in MsbA near the cytoplasmic leaflet of the plasma membrane was explored. Unlike the binding of core-LPS in the central cavity of MsbA, which has been identified as an intermediate state in the translocation cycle, the relevance of the core-LPS binding at the periphery of MsbA to the lipid translocation process is less clear. This PhD study utilised a wide range of methods, including cell biology, mutational analyses, biochemical assays, and molecular dynamics simulations to investigate the transport mechanisms of LmrA and MsbA. The findings enhance our understanding of the transport activities of bacterial multidrug ABC transporters, which may ultimately offer insights into the development of novel antibiotics that bypass or inhibit bacterial multidrug efflux pumps. As LmrA and MsbA are bacterial homologues of ABCB1, these insights could also contribute to the mechanistic studies of mammalian multidrug ABC transporters.","abstract_has_math":false,"creators":["Tang, Yakun"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Van Veen, Hendrik W"],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-10-31","date_published":"2023-10-31","updated_at":"2026-07-22T22:24:08Z","subjects":["ABC transporters","Antimicrobial resistance","Lipid transport","Membrane proteins","Multidrug efflux pumps"],"languages":["eng"],"rights":[],"rights_urls":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/29f74e07-116c-4ec4-ad5e-4614bd9828eb/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.109544","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Van Veen, Hendrik W"]},{"key":"dc:contributor.sponsor","label":"Sponsor","values":["The author was funded by the China Scholarship Council – Cambridge Trust PhD Scholarship"]},{"key":"dc:creator","label":"Author","values":["Tang, Yakun"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2023-10-31"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/369954"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["ABC transporters","Antimicrobial resistance","Lipid transport","Membrane proteins","Multidrug efflux pumps"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/29f74e07-116c-4ec4-ad5e-4614bd9828eb/download","https://www.rioxx.net/licenses/all-rights-reserved/"]},{"key":"dc:rights.embargodate","label":"Dc Rights Embargodate","values":["2026-06-26"]},{"key":"dc:rights.embargotype","label":"Dc Rights Embargotype","values":["embargo"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.109544"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/59eb9367-a9ae-427b-ad8e-e0fb12ed42d4/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["LmrA and MsbA are two paradigms in research on multidrug ATP-binding cassette (ABC) transporters. The bacterial transmembrane protein LmrA from *Lactococcus lactis* was the first known prokaryotic ABC transporter homologue of the mammalian multidrug resistance transporter ABCB1 (P-glycoprotein) characterised to have multidrug transport activity. A distinctive feature of LmrA as a primary-active transporter is its ability to harness electrochemical ion gradients. When measured in an electrophysiological setting, LmrA exhibits ion conductance upon the binding of ATP, which is based on a 2Na<sup>+</sup>/(1H<sup>+</sup>-1HEPES<sup>+</sup>-1Cl−)<sup>+</sup> exchange reaction. It also shows a Na<sup>+</sup>/ethidium<sup>+</sup> antiport reaction that can be driven by the sodium-motive force, which when directed inwardly in cells gives rise to enhanced ethidium efflux, and when directed outwardly in proteoliposomes gives rise to ethidium uptake by purified inside-out oriented LmrA. However, a detailed understanding of the sodium coupling mechanisms of LmrA is limited. LmrA has also been shown to mediate the transport of phospholipids, but the energetics of this process is unknown. To further examine the role of the sodium-motive force in the transport of a broader range of substrates, this PhD study investigated the transport of long-acyl-chain (2 x C18) headgroup biotin-labelled phosphatidylethanolamine (PE) by LmrA. In addition, the potential involvement of polar and acidic residues within LmrA in interaction with Na<sup>+</sup> was assessed. MsbA mediates the translocation of core lipopolysaccharides (core-LPS), hexa-acylated Lipid-A modified with the core oligosaccharides, as well as phospholipids across the plasma membrane in Gram-negative bacteria. It is essential for cell envelope integrity and has become an attractive target for the development of novel antibiotics against pathogenic bacteria. MsbA can also function as a multidrug transporter, sharing a broad substrate specificity for various drugs and cytotoxic agents with LmrA and ABCB1. Previously, using proteoliposomes containing purified MsbA and custom-made biotinylated-Lipid-A, the ability of MsbA to transport Lipid-A in an ATP-dependent fashion was biochemically demonstrated for the first time. Similar to the transport of small-molecule drugs, the flopping of physiologically relevant long-acyl-chain (2 x C18) headgroup biotin-labelled PE in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and a chemical proton gradient as sources of metabolic energy. The energetics of Lipid-A and PE transport was further investigated in this PhD study. Furthermore, the function of the recently reported peripheral (Kdo)2-Lipid-A (KDL) binding sites in MsbA near the cytoplasmic leaflet of the plasma membrane was explored. Unlike the binding of core-LPS in the central cavity of MsbA, which has been identified as an intermediate state in the translocation cycle, the relevance of the core-LPS binding at the periphery of MsbA to the lipid translocation process is less clear. This PhD study utilised a wide range of methods, including cell biology, mutational analyses, biochemical assays, and molecular dynamics simulations to investigate the transport mechanisms of LmrA and MsbA. The findings enhance our understanding of the transport activities of bacterial multidrug ABC transporters, which may ultimately offer insights into the development of novel antibiotics that bypass or inhibit bacterial multidrug efflux pumps. As LmrA and MsbA are bacterial homologues of ABCB1, these insights could also contribute to the mechanistic studies of mammalian multidrug ABC transporters."]},{"key":"dc:format.checksum.md5","label":"Dc Format Checksum Md5","values":["aa55fb07afdbe276ba1f991f96779e1e","87eda9de84448d1f82354d60eee3eb5f"]},{"key":"dc:title","label":"Title","values":["Investigations into the Lipid Transport Mechanisms and Energetics of Bacterial ATP-Binding Cassette (ABC) Transporters MsbA and LmrA"]}]}],"canonical_facts":{"dc:contributor.advisor":["Van Veen, Hendrik W"],"dc:contributor.sponsor":["The author was funded by the China Scholarship Council – Cambridge Trust PhD Scholarship"],"dc:creator":["Tang, Yakun"],"dc:date.issued":["2023-10-31"],"dc:description.abstract":["LmrA and MsbA are two paradigms in research on multidrug ATP-binding cassette (ABC) transporters. The bacterial transmembrane protein LmrA from *Lactococcus lactis* was the first known prokaryotic ABC transporter homologue of the mammalian multidrug resistance transporter ABCB1 (P-glycoprotein) characterised to have multidrug transport activity. A distinctive feature of LmrA as a primary-active transporter is its ability to harness electrochemical ion gradients. When measured in an electrophysiological setting, LmrA exhibits ion conductance upon the binding of ATP, which is based on a 2Na<sup>+</sup>/(1H<sup>+</sup>-1HEPES<sup>+</sup>-1Cl−)<sup>+</sup> exchange reaction. It also shows a Na<sup>+</sup>/ethidium<sup>+</sup> antiport reaction that can be driven by the sodium-motive force, which when directed inwardly in cells gives rise to enhanced ethidium efflux, and when directed outwardly in proteoliposomes gives rise to ethidium uptake by purified inside-out oriented LmrA. However, a detailed understanding of the sodium coupling mechanisms of LmrA is limited. LmrA has also been shown to mediate the transport of phospholipids, but the energetics of this process is unknown. To further examine the role of the sodium-motive force in the transport of a broader range of substrates, this PhD study investigated the transport of long-acyl-chain (2 x C18) headgroup biotin-labelled phosphatidylethanolamine (PE) by LmrA. In addition, the potential involvement of polar and acidic residues within LmrA in interaction with Na<sup>+</sup> was assessed. MsbA mediates the translocation of core lipopolysaccharides (core-LPS), hexa-acylated Lipid-A modified with the core oligosaccharides, as well as phospholipids across the plasma membrane in Gram-negative bacteria. It is essential for cell envelope integrity and has become an attractive target for the development of novel antibiotics against pathogenic bacteria. MsbA can also function as a multidrug transporter, sharing a broad substrate specificity for various drugs and cytotoxic agents with LmrA and ABCB1. Previously, using proteoliposomes containing purified MsbA and custom-made biotinylated-Lipid-A, the ability of MsbA to transport Lipid-A in an ATP-dependent fashion was biochemically demonstrated for the first time. Similar to the transport of small-molecule drugs, the flopping of physiologically relevant long-acyl-chain (2 x C18) headgroup biotin-labelled PE in proteoliposomes requires the simultaneous input of ATP binding and hydrolysis and a chemical proton gradient as sources of metabolic energy. The energetics of Lipid-A and PE transport was further investigated in this PhD study. Furthermore, the function of the recently reported peripheral (Kdo)2-Lipid-A (KDL) binding sites in MsbA near the cytoplasmic leaflet of the plasma membrane was explored. Unlike the binding of core-LPS in the central cavity of MsbA, which has been identified as an intermediate state in the translocation cycle, the relevance of the core-LPS binding at the periphery of MsbA to the lipid translocation process is less clear. This PhD study utilised a wide range of methods, including cell biology, mutational analyses, biochemical assays, and molecular dynamics simulations to investigate the transport mechanisms of LmrA and MsbA. The findings enhance our understanding of the transport activities of bacterial multidrug ABC transporters, which may ultimately offer insights into the development of novel antibiotics that bypass or inhibit bacterial multidrug efflux pumps. As LmrA and MsbA are bacterial homologues of ABCB1, these insights could also contribute to the mechanistic studies of mammalian multidrug ABC transporters."],"dc:format.checksum.md5":["aa55fb07afdbe276ba1f991f96779e1e","87eda9de84448d1f82354d60eee3eb5f"],"dc:identifier.doi":["https://doi.org/10.17863/CAM.109544"],"dc:identifier.uri":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/59eb9367-a9ae-427b-ad8e-e0fb12ed42d4/download"],"dc:language":["eng"],"dc:publisher.institution":["University of Cambridge"],"dc:relation.isreferencedby.uri":["https://www.repository.cam.ac.uk/handle/1810/369954"],"dc:rights":["https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/29f74e07-116c-4ec4-ad5e-4614bd9828eb/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"dc:rights.embargodate":["2026-06-26"],"dc:rights.embargotype":["embargo"],"dc:subject":["ABC transporters","Antimicrobial resistance","Lipid transport","Membrane proteins","Multidrug efflux pumps"],"dc:title":["Investigations into the Lipid Transport Mechanisms and Energetics of Bacterial ATP-Binding Cassette (ABC) Transporters MsbA and LmrA"],"dc:type":["Thesis"],"dc:type.qualificationlevel":["Doctoral"],"dc:type.qualificationname":["Doctor of Philosophy (PhD)"]},"updated_at":"2026-07-22T22:24:08Z"}