Structural basis of ion - substrate coupling in the Na+-dependent dicarboxylate transporter VcINDY

Abstract

The Na⁺-dependent dicarboxylate transporter from Vibrio cholerae (VcINDY) is a prototype for the divalent anion sodium symporter (DASS) family. While the utilization of an electrochemical Na⁺ gradient to power substrate transport is well established for VcINDY, the structural basis of this coupling between sodium and substrate binding is not currently understood. Here, using a combination of cryo-EM structure determination, succinate binding and site-directed cysteine alkylation assays, we demonstrate that the VcINDY protein couples sodium- and substrate-binding via a previously unseen cooperative mechanism by conformational selection. In the absence of sodium, substrate binding is abolished, with the succinate binding regions exhibiting increased flexibility, including HP_inb, TM10b and the substrate clamshell motifs. Upon sodium binding, these regions become structurally ordered and create a proper binding site for the substrate. Taken together, these results provide strong evidence that VcINDY's conformational selection mechanism is a result of the sodium-dependent formation of the substrate binding site.

Fig. 1. Cryo-EM structure of VcINDY in the C_i-Na⁺ state determined in 300 mM Na⁺.

a Measurements of succinate binding to detergent-purified VcINDY in the presence of 100 mM NaCl, using intrinsic tryptophan fluorescence quenching (N = 4). Data are presented as mean values ± SEM. The apparent K_d was determined to be 92.2 ± 47.4 mM. When NaCl was replaced with Choline chloride, no binding of succinate to VcINDY could be measured (N = 4). b Cryo-EM map of VcINDY determined in the presence of 300 mM NaCl. The map is colored by local resolution (Å) and contoured at 5.1 σ. The overall map resolution is 2.83 Å. c Structure of VcINDY in the C_i-Na⁺ state. The structure is colored by the B-factor. d Na1 site structure and Coulomb map. e Na2 site structure and Coulomb map. f Overlay of VcINDY structures around the substrate and sodium binding sites in the C_i-Na⁺ state (green) and C_i-Na⁺-S state (PDB ID: 5UL7, blue). There is very little structural change observed between the two states.

Fig. 2. Cryo-EM structure of VcINDY in the C_i-apo state determined in Choline⁺.

a Cryo-EM map of VcINDY preserved in amphipol determined in the presence of 100 mM Choline Chloride. The map is colored by local resolution (Å) on the same scale as Fig. 1b and contoured at 4.8 σ. The overall map resolution is 3.23 Å. The two previously-identified hinge regions which facilitate movement of the transport domain, L4-HP_in and L9-HP_out, are found to be most flexible. b Structure of VcINDY in the C_i-apo state. The structure is colored by the B-factor on the same scale as Fig. 1b. c Overlay of VcINDY structures around substrate and sodium binding sites in the sodium-bound C_i-Na⁺ state (green) and the C_i-apo state (pink). The structures of the two Na1 and Na2 clamshells have changed in the absence of sodium ions, particularly around residues Ile149, Asn151, Ala376, Asn378, Ala420 and Pro422.

Fig. 3. VcINDY flexibility changes near the Na1 and Na2 sites between the C_i-Na⁺ state and C_i-apo states.

a Cryo-EM density map in 300 mM NaCl. b. Cryo-EM density map in 100 mM Choline Chloride. In a and b, the respective protein models’ backbones are fitted into the densities. Maps are contoured such that the scaffold domains have equal volume. c. Structure of VcINDY in its C_i-Na⁺ state. d. Structure of VcINDY in its C_i-apo state. In c and d, the structures are colored by normalized B-factors. e NMR-style analysis of the VcINDY structure in Na⁺. f NMR-style analysis of the VcINDY structure in Choline⁺. The resolution for refinement of both structures in e and f was truncated to 3.23 Å. In the absence of sodium, the helices on the cytosolic side of Na1 and Na2, particularly HP_inb and TM10b and their connecting loops, show markedly increase flexibility. Instead of a single structure, the C_i-apo model consists of an ensemble of structures.

Fig. 4. Cysteine alkylation with mPEG5K of VcINDY near the Na1 and Na2 sites in the presence and absence of Na⁺.

a Location of cysteine mutations. Our structures suggested that HP_inb and TM10b become flexible in the absence of sodium, increasing the solvent accessibility of Leu138, Ala155, Val162 and Ala189 near the Na1 site, and Val441 near the Na2 site. Position Ser436, for which no accessibility change was observed between our structures, is used as a control. On a Cys-less background, residues at these positions were individually mutated to a cysteine for mPEG5K labeling. For clarity, only amino acid numbers are labeled and the types are omitted. b Coomassie Brilliant Blue-stained non-reducing polyacrylamide gels showing the site-directed PEGylation of each cysteine mutant over time in the presence and absence of Na⁺. P: PEGylated protein; U: Un-PEGylated protein. Each reaction was performed on two separate occasions with the same result. Source data is provided as Source Data file.

Fig. 5. Movement of VcINDY’s amino acid side chains between its C_i-Na⁺-S, C_i-Na⁺ and C_i-apo states.

VcINDY structures in three states are overlaid: C_i-Na⁺-S (blue), C_i-Na⁺ (green) and C_i-apo (pink) states. a At the scaffold-transport domain interface, the side chains of Phe100, His111 and Phe326 rotate between states. b On the periplasmic surface, some loops and side chains move between the states, including Phe220, Lys337, Glu394 and Trp462.

Fig. 6. Schematic model of conformational selection mechanism for sodium—substrate coupling in VcINDY.

In the absence of sodium ions, HP_inb and TM10b, along with their connecting loops responsible for sodium and substrate binding, are flexible. From the ensemble of flexible structures, the binding of sodium ions (blue circles) selects a conformation with a proper binding site for the substrate, allowing its binding (red oval). The scaffold and transport domains in each protomer are colored as green and pink, respectively. Only the Na1 and Na2 sites are illustrated. Transport domain movements in the two protomers are shown as symmetric for simplicity but are functionally independent.