Iodide Binding in Sodium-Coupled Cotransporters

Abstract

Several apical iodide translocation pathways have been proposed for iodide efflux out of thyroid follicular cells, including a pathway mediated by the sodium-coupled monocarboxylate transporter 1 (SMCT1), which remains controversial. Herein, we evaluate structural and functional similarities between SMCT1 and the well-studied sodium-iodide symporter (NIS) that mediates the first step of iodide entry into the thyroid. Free-energy calculations using a force field with electronic polarizability verify the presence of a conserved iodide-binding pocket between the TM2, TM3, and TM7 segments in hNIS, where iodide is coordinated by Phe67, Gln72, Cys91, and Gln94. We demonstrate the mutation of residue Gly93 of hNIS to a larger amino acid expels the side chain of a critical tryptophan residue (Trp255) into the interior of the binding pocket, partially occluding the iodide binding site and reducing iodide affinity, which is consistent with previous reports associating mutation of this residue with iodide uptake deficiency and hypothyroidism. Furthermore, we find that the position of Trp255 in this hNIS mutant mirrors that of Trp253 in wild-type hSMCT1, where a threonine (Thr91) occupies the position homologous to that occupied by glycine in wild-type hNIS (Gly93). Correspondingly, mutation of Thr91 to glycine in hSMCT1 makes the pocket structure more like that of wild-type hNIS, increasing its iodide affinity. These results suggest that wild-type hSMCT1 in the inward-facing conformation may bind iodide only very weakly, which may have implications for its ability to transport iodide.

Figure 1

Structural modeling of hSMCT1 and hNIS using the vSGLT transporter as a template. (A) Schematic representation of hSMCT1/hNIS topology with the N- and C-terminus exposed to the extracellular medium and cytoplasm, respectively. The helices on the red triangular background comprise repeat 1, while repeat 2 is composed of the helices on the blue triangular background. (B) Helix representation of the hSMCT1 model, rendered with Bendix, in an inward-facing conformation viewed from the plane of the membrane, with the extracellular side at the top. The helices are colored according to the topology with triangular backgrounds indicating the orientation of repeats. The position of the protein in the membrane was defined after its superposition onto that of vSGLT, which was determined with the OPM server. The hNIS model shows a similar fold. (C) Ensemble of the refined sequence alignments (vSGLT/hNIS and vSGLT/hSMCT1) used for modeling. The alignment is colored according to the chemical properties of the residues: gray, aliphatic (A, I, L, M, and V); cyan, polar uncharged (N, Q, S, and T); yellow, aromatic (F, W, and Y); red, acidic (D and E); purple, basic (K, R, and H); pink, exceptional (C, G and P). The secondary structure (helix) assignment for the vSGLT crystal structure was obtained with DSSP and is indicated by dark blue rectangles.

Figure 2

Predicted sodium-binding sites in hNIS and hSMCT1 and the conservation of associated residues. After superimposing the models on the vSGLT crystal structure, a putative sodium-binding site was predicted for (A) hNIS and (B) hSMCT1. The sodium ion reported in vSGLT, used as a reference point for our analysis, is shown as a purple sphere. The predicted sodium-binding site conserved in both models involves residues in TM2, TM6, TM7, and TM9 segments, which are shown in a cartoon representation and colored as in Figure 1. The putative sodium-coordinating residues are displayed as sticks colored by atom type (oxygen in red and nitrogen in blue). The participation of residues S66, D191, Q194, and Q263 in hNIS-mediated iodide uptake has been recently demonstrated by experiments. The hNIS and hSMCT1 models are oriented with the extracellular side toward the top of the page. (C) Sequence logo illustrating conservation of the residues (indicated with arrows) predicted to coordinate the Na⁺ ion. The conservation was evaluated among 405 members of the SSS family. The residue colors are as follows: polar (G, S, T, Y, and C) in green, amide-terminated (N and Q) in purple, basic (K, R, and H) in blue, acidic (D and E) in red, and hydrophobic (A, V, L, I, P, W, F, and M) in black. Numbering follows the sequence of hNIS.

Figure 3

Predicted iodide-binding pockets in hNIS and hSMCT1 and the conservation of associated residues. For the (A) hNIS and (B) hSMCT1 models, these sites involve the transmembrane TM2, TM3, and TM7 segments, which are shown in a cartoon representation and colored as in Figure 1. The nine putative residues coordinating iodide in both models are displayed as sticks colored by atom type (oxygen in red, nitrogen in blue, and sulfur in light yellow). Residues G93/T91 are highlighted in blue. G93 in hNIS was found to be mutated in patients with goitrous hypothyroidism. The galactose (Gal) molecule reported in vSGLT is located near the predicted iodide position in the models and is shown here for reference as transparent blue and red sticks. The predicted iodide binding cavities are displayed with the extracellular side toward the top of the page. (C) Sequence alignment between hNIS and hSMCT1 showing the conservation of residues proposed to coordinate iodide. The alignment is colored according to the chemical properties of the residues as in Figure 1. The secondary structure assignments obtained with PSIPRED (helix) are indicated by dark blue rectangles. Arrows indicate the residues involved in iodide binding according to our prediction.

Figure 4

Free-energy calculation of iodide binding to hNIS and hSMCT1. (A) Image showing the putative binding site and proposed iodide exit pathway through hNIS, indicating their position relative to the complete protein. (B–E) Magnification of the binding site and iodide entry/exit pathway in wild-type and mutant hNIS and hSMCT1. The 3D free-energy map is represented by purple surfaces, where regions with free energies < −1 and < −3 kcal/mol relative to the value at a reference position are enclosed by transparent and solid surfaces, respectively. The C atoms of the W255/253 are green, and those of residue 93/91 are orange. The residues with C atoms in cyan are those within 4.5 Å of the minimum free energy.

Figure 5

Role of residue 93 of hNIS and the homologous residue 91 of hSMCT1 in iodide binding. (A) In this simulation snapshot of hNIS WT, the tryptophan (W255) forms the wall of the putative iodide-binding pocket, making contact with G93. (B) In the hNIS G93T mutant, the steric bulk of the T93 side chain expels W255 into the pocket. (C) Histogram of the distance between the ring nitrogen of W255 (W253 in hSMCT1) and the C_α atom of the residue at position 93 (91 in hSMCT1).

Figure 6

Free energy calculations on fragments of wild-type hNIS, hSMCT1, and their mutants. (A) Structure of the fragment. The portion of the hNIS WT included in the fragment is shown in color (TM2, red; TM3, orange; TM7 green; TM11, blue) by broad ribbons, while the part not included is shown by thin gray ribbons. The position of lowest free energy for iodide is represented by a purple sphere, while the region to which the iodide was restrained during the calculation is shown as a transparent purple cylinder. (B) Free energy as a function of the position of iodide along the axis of the cylinder shown in panel A.