Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters.

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neurotransmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and liver. We combined classical molecular dynamics simulations, Markov state modeling and hybrid first principles quantum mechanical/classical mechanical (QM/MM) simulations with experimental approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton transfer from D46 to E133 opens the intracellular gate and permits galactonate dissociation either in its unprotonated form or after proton transfer from E133. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.