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.