Energetics and mechanisms of substrate transport by the MATE transporter PfMATE

Abstract

Multidrug resistance of microbial pathogens and cancers is a significant global challenge, imposing substantial economic and social burdens. Organisms ranging from bacteria to mammals can develop resistance to previously effective drugs, which compromises the therapeutic outcomes. The misuse and over-prescription of antimicrobials contribute significantly to this problem, undermining the efficacy of treatments for routine infections and compromising the success of critical medical interventions. Additionally, the widespread occurrence of multidrug resistant pathogens has raised alarms due to their severe consequences, which include higher rates of treatment failures, prolonged hospital stays, and increased mortality. Multidrug transporters play a critical role in the multidrug resistance. These efflux pumps can use the energy derived from ATP hydrolysis and/or electrochemical ion gradients to actively remove structurally unrelated drugs, such as antimicrobials and anticancer drugs, from cells. PfMATE, a MATE (multidrug and toxic compound extrusion) transporter, has gained wide attention for being one of the best characterised ion-coupled multidrug transporters with both outward-facing and inward-facing protein conformations determined. Although advanced techniques have brought insights into the structural features of PfMATE, the intricate details of its transport mechanism, including substrate selection, ion coupling, ion-drug stoichiometry, and the role of lipids, have remained elusive. In this PhD project, I employed lactococcal cells to measure ethidium transport by PfMATE. This method involved the use of various ionophores to manipulate the composition and magnitude of proton motive forces across the plasma membrane of these cells. My findings indicate that PfMATE mediates electroneutral ethidium+ /H + antiport independent of membrane potential. Alongside with mutagenesis work, this investigation not only identified the essential carboxylates integral to PfMATE activity, but also revealed an alternative ion translocation pathway that can sustain PfMATE operational integrity when one pathway is compromised. Subsequently, this project focused on a detailed exploration of the energetics and the roles of key residues in ethidium transport process using proteoliposomes containing purified PfMATE protein. This advanced approach allows for precise manipulation of experimental parameters, particularly ion motive forces, and creates an artificial lipid environment for PfMATE. I discovered that PfMATE can recognise both H + and Na + as a coupling ion and that it can catalyse the electroneutral exchange of ethidium with either of these ions, equivalent to ethidium+ /(1H + or 1Na+ ) antiport. Critical residues from both N- and C-lobes of PfMATE were identified that participate in the relevant ion translocation pathways. Lastly, this project investigated the lipid transport activity of PfMATE. Prior molecular dynamics simulations proposed the entry and reorientation of lipids within the positively charged central cavity of PfMATE. In biochemical lipid transport assays, I tested this hypothesis and demonstrated that PfMATE functions as a lipid floppase by facilitating the translocation of phosphatidylethanolamine from one leaflet to the other leaflet of the phospholipid bilayer. This process uniquely depends on the chemical Na + gradient while being independent of the chemical H + gradient. In summary, my thesis presents a comprehensive study of PfMATE, employing functional assays to unravel its transport mechanisms for both ethidium and lipids. The findings not only advance our understanding of the mechanistic diversity of PfMATE but also contribute valuable insights to the broader field of the MATE transporter family.