Mapping of Ion and Substrate Binding Sites in Human Sodium Iodide Symporter (hNIS)

Abstract

The human sodium iodide symporter (hNIS) is a theranostic reporter gene which concentrates several clinically approved SPECT and PET radiotracers and plays an essential role for the synthesis of thyroid hormones as an iodide transporter in the thyroid gland. Development of hNIS mutants which could enhance translocation of the desired imaging ions is currently underway. Unfortunately, it is hindered by lack of understanding of the 3D organization of hNIS and its relation to anion transport. There are no known crystal structures of hNIS in any of its conformational states. Homology modeling can be very effective in such situations; however, the low sequence identity between hNIS and relevant secondary transporters with available experimental structures makes the choice of a template and the generation of 3D models nontrivial. Here, we report a combined application of homology modeling and molecular dynamics refining of the hNIS structure in its semioccluded state. The modeling was based on templates from the LeuT-fold protein family and was done with emphasis on the refinement of the substrate-ion binding pocket. The consensus model developed in this work is compared to available biophysical and biochemical experimental data for a number of different LeuT-fold proteins. Some functionally important residues contributing to the formation of putative binding sites and permeation pathways for the cotransported Na⁺ ions and I^- substrate were identified. The model predictions were experimentally tested by generation of mutant versions of hNIS and measurement of relative (to WT hNIS) ¹²⁵I^- uptake of 35 hNIS variants.

Figure 1.

Comparison of three different 3D hNIS models built with protein templates from the chloride transporter CLC-ec1 (PDB: 1KPK), the bicarbonate transporters hAE1 (PDB: 4YZF) and hNBCe1 (PDB: 6CAA) from the SLC4 family, and the LeuT-fold proteins vSGLT (PDB: 3DH4) and Mhp1 (PDB: 4D1B). The position of acidic and basic residues is indicated with red and blue colors, respectively. Key residues for iodide uptake identified in hNIS from functional mutagenesis are shown as yellow spheres.

Figure 2.

(A) 3D model of hNIS based on the vSGLT and Mhp1 templates. The positions of the bound Na⁺ ions in the putative sites Na1 and Na2 (red spheres) and substrates (blue sticks) in the available crystal structures of LeuT, vSGLT, and Mhp1 are shown overlapped with the 3D model of hNIS. (B) Putative binding sites for cations (red spheres) and anionic substrates (blue spheres) in hNIS determined from combination of GCMC/PB calculations.

Figure 3.

Average ¹²⁵I⁻ relative uptake of selected hNIS mutants studied in the present work, calculated as fractions of the uptake of cells expressing WT hNIS. Values below 1 signify a decrease in ¹²⁵I⁻ uptake compared to WT hNIS.

Figure 4.

Tentative Na2 binding site for Na⁺ (magenta sphere) in hNIS, based on the contact frequency maps in Figures S3 and S4, the ion density map in Figure S5, and the residues listed in Table S2.

Figure 5.

Tentative S1 and S2 binding sites for I⁻ (purple sphere) in hNIS based on the contact frequency maps in Figures S3 and S4, the I⁻ density map in Figure S5, and the residues listed in Table S2. The residues are color coded: green for residues involved in both sites S1 and S2, dark blue for residues unique to site S1, and cyan for residues unique to site S2.

Figure 6.

Water density map calculated from 13 MD simulations of hNIS with 2:1 Na⁺:I⁻ ion stoichiometry (no water in binding cavity at MD onset). The I⁻ ion is shown as a purple sphere and is located in the S1 site. The S1 site is accessible to water from the intracellular space, consistent with the inward-facing open state of hNIS. The residues shown as magenta sticks line the water permeation pathway. Residues from the hydrophobic constriction zone at the extracellular side of hNIS are shown as pink sticks. The G425-P426 motif, responsible for opening of the extracellular gate is shown as red spheres.