Structural Dynamics of Active Transporters - The long-term goal of the PI’s research program is to illuminate the structure, dynamics and mechanistic principles that underpin active efflux of solutes, often of impressive size such as protein domains, across cell membranes. Clinical multidrug resistance (MDR) in the treatment of bacterial and fungal infections, and chemotherapy of neoplasms can be associated with overexpression of membrane-embedded efflux pumps, collectively referred to as MDR transporters, that selectively extrude cytotoxic molecules from the cell. MDR transporters harness the free energy of ATP hydrolysis or that stored in electrochemical gradients to power a conformational cycle that drives the energetically uphill vectorial translocation of substrates. The cycle entails the energy-coupled isomerization of the transporter between multiple intermediates thereby executing alternating access of the substrate binding site. Defining the structural elements mediating alternating access and decoding the mechanism of energy conversion in a lipid bilayer environment are central questions in the field and critical for elucidating transport mechanisms. This MIRA proposal will continue support of two established, productive research programs focused on addressing these questions for ATP binding cassette (ABC) and Multidrug and Toxin Extrusion (MATE) transporters. Our innovative experimental blueprint capitalizes on recent transformational advances in machine learning protein structure prediction, state of the art electron paramagnetic spectroscopy (EPR) tools in the context of high resolution cryoEM structures. Project 1, motivated and grounded in the contribution of the PI’s group, seeks to decipher ion-substrate coupling, to define conserved and divergent elements of alternating access, and to reveal specific transporter-lipids interactions that shape the energy landscape of conformational changes in MATE transporters. Project 2 will expand a long-standing investigation of energy transduction and alternating access ABC efflux transporters in three archetypes that represent a spectrum of energy conversion and substrate size. The two projects will illuminate mechanistic principles for families of transporters implicated in the phenomena of drug resistance and basic bacterial defense strategies.
