Functional role of a structural water in the elevator domain of dicarboxylate transporter VcINDY

The divalent anion sodium symporter (DASS) family mediates the uptake of Krebs cycle intermediates and sulfate and influences adiposity, insulin resistance, and metabolism in mammals. While Na⁺:substrate stoichiometry is known for several DASS transporters, the location of key Na⁺-binding sites remains elusive; important information for understanding the mechanism. In VcINDY, a bacterial DASS protein, we visualized a nonprotein cryo-electron microscopy (cryo-EM) density in the middle of the transport domain. Its size and coordination suggest that it may represent either a third Na⁺ ion or a structural water molecule. Using a combination of in vitro binding and transport assays, cryo-EM structural determination, and molecular dynamic simulations, we show that the density is not a Na⁺ ion. Instead, the data indicate that the density likely represents a structural water molecule critical for transport domain integrity. Sequence and structural similarities suggest this feature may be conserved across human DASS transporters such as NaCT and NaDC3.