Structure and mechanism of a bacterial sodium-dependent dicarboxylate transporter

Abstract

In human cells, cytosolic citrate is a chief precursor for the synthesis of fatty acids, triacylglycerols, cholesterol and low-density lipoprotein. Cytosolic citrate further regulates the energy balance of the cell by activating the fatty-acid-synthesis pathway while downregulating both the glycolysis and fatty-acid β-oxidation pathways. The rate of fatty-acid synthesis in liver and adipose cells, the two main tissue types for such synthesis, correlates directly with the concentration of citrate in the cytosol, with the cytosolic citrate concentration partially depending on direct import across the plasma membrane through the Na(+)-dependent citrate transporter (NaCT). Mutations of the homologous fly gene (Indy; I'm not dead yet) result in reduced fat storage through calorie restriction. More recently, Nact (also known as Slc13a5)-knockout mice have been found to have increased hepatic mitochondrial biogenesis, higher lipid oxidation and energy expenditure, and reduced lipogenesis, which taken together protect the mice from obesity and insulin resistance. To understand the transport mechanism of NaCT and INDY proteins, here we report the 3.2 Å crystal structure of a bacterial INDY homologue. One citrate molecule and one sodium ion are bound per protein, and their binding sites are defined by conserved amino acid motifs, forming the structural basis for understanding the specificity of the transporter. Comparison of the structures of the two symmetrical halves of the transporter suggests conformational changes that propel substrate translocation.

Fig. 1

Functional characterization and structure determination of the Na⁺-dependent dicarboxylate transporter vcINDY from Vibrio cholerae. a, Na⁺-driven succinate transport by vcINDY measured in whole-cells ^,. The succinate uptake was measured in vcINDY-transformed E. coli in buffers that contained 5 μM [¹⁴C]succinate and either Na⁺, Li⁺ or K⁺. Thecontrol experiment was carried out in Na⁺ buffer using cells that were transformed with empty vector. b, Uptake of [¹⁴C]succinate in the presence of various di- and tri-carboxylates and sulfate (at 1 mM concentration). For a and b, N = 3. c, Crystal structure of the vcINDY dimer at 3.2 Å resolution viewed from within the membrane. A citrate and a Na⁺ ion are adjacently bound to each vcINDY protomer at the cytosolic basin of the protein dimer. d, Crystal structure of the vcINDY dimer viewed from the cytosol. The bound citrate is exposed to the cytosolic space whereas the Na⁺ ion is buried. In c and d, the polypeptide in one protomer is colored using the standard rainbow scheme.

Fig. 2

Structure of the vcINDY protomer. a, Transmembrane topology of vcINDY. The two halves of the protein, TMs2-6 and TMs7-11, are related by a repeat in amino acid sequence, resulting in a transmembrane topology that displays an inverted twofold symmetry. b, The N-and C-terminal halves of the protomer each forms a hand-shaped structure, and the two hands are related by an inverted twofold symmetry. TMs2&3 form the thumb, and the helical bundle of TM4b – TM6 takes the shape of the palm in the N-terminal half; in the C-terminal half, the thumb is formed by TMs7&8 and the palm by TM9a – TM11. Note the linker helix between the palm and the thumb in the N-terminal half is at a larger angle from the membrane plane than that of the linker in the C-terminal hand, giving the former a V-shape and the latter a U-shape. The structures of two helical bundles, the palms, are similar and their superposition yields an r.m.s.d. of 2.9 Å for backbone Cα atoms. The N- and C-terminal halves of the protein are colored green and purple, respectively.

Fig. 3

Na⁺ ion-binding sites in vcINDY. a, Structure of the Na⁺-binding site (Na1) formed by the tip of HP_in and the L5ab loop. The binding site has the shape of a clamshell, which we named the “hairpin tip – capping loop motif” for sodium binding. A second, putative Na⁺-binding site (Na2), is suggested to be located between the tip of HP_out and the L10ab loop formed by the C-terminal hairpin tip – capping loop motif. However, no electron density for Na⁺ was found at this site in the crystal structure. In the current inward-facing transporter structure, the Na2 site is directly exposed to the cytosolic space. b, Coordination of the Na⁺ ion at the Na1 site. Both side chains of amino acid residues and backbone carboxyl oxygen atoms are involved in the Na⁺ coordination. c, Succinate transport activity of Na1-site mutants. N = 3.

Fig. 4

Substrate-binding site in vcINDY. a, Electrostatic surface potential of the substrate-binding site. Insert: cross section of the electrostatic surface potential of vcINDY dimer. The plane of this central cross section is perpendicular to the membrane and is at a small angle from the long axis of the dimer in order to show both citrate molecules bound to the transporter dimer. The arrow points in the direction for the view in a. b, Structure of the substrate-binding site with a citrate bound, showing the coordination of the substrate analog. Three hydrogen bonds are indicated by dashed lines. The citrate lies at a small angle to the membrane plane, and its long axis is parallel to the protomer-protomer interface. The central 6-hydroxyl-, carboxyl-groups are exposed to the cytosolic space. While the side chain of Ser150 forms a hydrogen bond with the 5-carboxyl group of the citrate, its backbone carbonyl oxygen atom participates in the coordination of the Na1 ion. Similarly, the side chain of Asn151 interacts with both Na1 and the bound citrate. c, Amino acid sequence alignment of vcINDY and its homologs, showing the two SNT carboxylate-binding motifs. d, Succinate transport activity of substrate-binding site mutants. N = 3.