Determinants of substrate and cation transport in the human Na+/dicarboxylate cotransporter NaDC3.

Metabolic intermediates, such as succinate and citrate, regulate important processes ranging from energy metabolism to fatty acid synthesis. Cytosolic concentrations of these metabolites are controlled, in part, by members of the SLC13 gene family. The molecular mechanism underlying Na(+)-coupled di- and tricarboxylate transport by this family is understood poorly. The human Na(+)/dicarboxylate cotransporter NaDC3 (SLC13A3) is found in various tissues, including the kidney, liver, and brain. In addition to citric acid cycle intermediates such as α-ketoglutarate and succinate, NaDC3 transports other compounds into cells, including N-acetyl aspartate, mercaptosuccinate, and glutathione, in keeping with its dual roles in cell nutrition and detoxification. In this study, we construct a homology structural model of NaDC3 on the basis of the structure of the Vibrio cholerae homolog vcINDY. Our computations are followed by experimental testing of the predicted NaDC3 structure and mode of interaction with various substrates. The results of this study show that the substrate and cation binding domains of NaDC3 are composed of residues in the opposing hairpin loops and unwound portions of adjacent helices. Furthermore, these results provide a possible explanation for the differential substrate specificity among dicarboxylate transporters that underpin their diverse biological roles in metabolism and detoxification. The structural model of NaDC3 provides a framework for understanding substrate selectivity and the Na(+)-coupled anion transport mechanism by the human SLC13 family and other key solute carrier transporters.

The ups and downs of elevator-type di-/tricarboxylate membrane transporters.

The divalent anion sodium symporter (DASS) family contains both sodium-driven anion cotransporters and anion/anion exchangers. The family belongs to a broader ion transporter superfamily (ITS), which comprises 24 families of transporters, including those of AbgT antibiotic efflux transporters. The human proteins in the DASS family play major physiological roles and are drug targets. We recently determined multiple structures of the human sodium-dependent citrate transporter (NaCT) and the succinate/dicarboxylate transporter from Lactobacillus acidophilus (LaINDY). Structures of both proteins show high degrees of structural similarity to the previously determined VcINDY fold. Conservation between these DASS protein structures and those from the AbgT family indicates that the VcINDY fold represents the overall protein structure for the entire ITS. The new structures of NaCT and LaINDY are captured in the inward- or outward-facing conformations, respectively. The domain arrangements in these structures agree with a rigid body elevator-type transport mechanism for substrate translocation across the membrane. Two separate NaCT structures in complex with a substrate or an inhibitor allowed us to explain the inhibition mechanism and propose a detailed classification scheme for grouping disease-causing mutations in the human protein. Structural understanding of multiple kinetic states of DASS proteins is a first step toward the detailed characterization of their entire transport cycle.