PROJECT SUMMARY The plasma membrane-bound sodium-dependent citrate transporter (NaCT) plays a major role in fatty acid biosynthesis and thus represents a major therapeutic target for various metabolic diseases. Its bacterial homolog, VcINDY, is a prototype of the entire divalent anion sodium symporter (DASS) family, of which NaCT is also a member. Despite the enormous progress that has been made, major gaps remain in our current understanding of the molecular mechanisms of NaCT, VcINDY, and other secondary membrane transporters. In particular, we do not know how a transporter works in real-time. At best, mechanistic descriptions of the transport cycles of these transporters consist of a series of structural snapshots of some of the individual states, along with limited kinetic parameters connecting the various states obtained from ensemble measurements. In this project, we aim to characterize the structure, dynamics, function, and inhibition of these two sodium-driven carboxylate transporters. (1) We will first characterize the structural basis of sodium-substrate coupling in VcINDY and NaCT. We will identify the ion stoichiometry and the positions and structures of the unknown sodium sites in the transporters. Following measurements of sodium-dependent substrate binding in solution, we will determine the structures of the transporters in the presence and absence of sodium using cryo-EM. Any hypothesis suggested by such structures will be examined by biochemical experiments. (2) Using smFRET, we will characterize the real-time dynamics with which VcINDY and NaCT transition between the outward- and inward-facing states during transport. Specifically, we will identify the number of conformational states that are sampled during transport and determine the rates of transitions between those states. Using this framework, we will investigate how the sodium ions and substrates that generate the driving force for transport modulate these parameters. These experimental measurements will be integrated into computational models generated by MD simulations. (3) We will aim to understand the entire reaction cycle of the transporter, including the energy landscape. We will develop FRET-based succinate and citrate sensors to measure the transport activities of individual transporters in single liposomes. These measurements will be used to construct the entire free-energy landscape of the transporter using MD simulations. (4) Finally, we will elucidate the mechanisms of inhibition of NaCT by several classes of allosteric inhibitors from the pharmaceutical industry to improve their potency and specificity in order to help design better strategies in the treatment of metabolic disorders.

NARRATIVE The sodium-dependent citrate transporter NaCT plays a key role in fatty acid biosynthesis. In this multi-PI R01, we will combine structural (cryo-EM), dynamic (single molecule FRET and molecular dynamics simulations) and kinetic (transport and binding assays) techniques to understand the transport mechanism of NaCT and its bacterial homolog VcINDY. The information obtained will help in understanding the molecular pathogenesis of various metabolic diseases such as diabetes, obesity and cancer, and may aid in the development of potential treatments.