Abstract
Citrate, α-ketoglutarate and succinate are TCA cycle intermediates that also play essential roles in metabolic signaling and cellular regulation. These di- and tricarboxylates are imported into the cell by the divalent anion sodium symporter (DASS) family of plasma membrane transporters, which contains both cotransporters and exchangers. While DASS proteins transport substrates via an elevator mechanism, to date structures are only available for a single DASS cotransporter protein in a substrate-bound, inward-facing state. We report multiple cryo-EM and X-ray structures in four different states, including three hitherto unseen states, along with molecular dynamics simulations, of both a cotransporter and an exchanger. Comparison of these outward- and inward-facing structures reveal how the transport domain translates and rotates within the framework of the scaffold domain through the transport cycle. Additionally, we propose that DASS transporters ensure substrate coupling by a charge-compensation mechanism, and by structural changes upon substrate release.
Highlights
Citrate and dicarboxylates such as a-ketoglutarate, succinate and malate are intermediates of the TCA cycle
We aimed to characterize the structural basis of the entire transport cycle of the divalent anion sodium symporter (DASS) family using a combination of single particle cryo-electron microscopy (cryo-EM), X-ray crystallography, molecular dynamics (MD) simulations and transport activity assays
The total mass of a VcINDY dimer is only 96 kDa, and almost that entire mass is embedded in the membrane
Summary
Citrate and dicarboxylates such as a-ketoglutarate (aKG), succinate and malate are intermediates of the TCA cycle. These molecules play essential roles in metabolic signaling and cellular regulation (Tannahill et al, 2013; Mills et al, 2018; Huergo and Dixon, 2015). Cytoplasmic aKG and succinate are important in controlling cell fate. Naive embryonic stem cells that exhibit an elevated aKG-to-succinate ratio maintain pluripotency (Carey et al, 2015). Pancreatic ductal adenocarcinoma cells with p53-deficiency have a lowed aKG-to-succinate ratio, and increasing the cellular concentration of aKG leads to a phenotype similar to that of tumor suppression by p53 restoration (Morris et al, 2019)
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