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.

Structural insights into the mechanism of the sodium/iodide symporter (NIS)

“…2d). Similar results have been reported with substitutions at some of these positions in human NIS 28 . Here we show that the effects of the replacements were clearly not due to impaired NIS mutant protein expression or trafficking to the cell surface, because all mutants were present at the plasma membrane at levels comparable to those of WT NIS, as determined by flow cytometry with an antibody against the extracellularly facing HA tag (Extended Data Fig.…”

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism

“…A natural solution to this challenge is to combine transport measurements, protein sequence comparisons, and/or structural and molecular modeling ( 33 , 34 ) using other protein families or within the SLC4 family. This comprehensive experiment-modeling approach aids in the targeted validation of the putative binding pockets and establishes protein–ion/substrate interactions of specific functionally critical amino acid residues ( 35 , 36 ).…”

Sodium/Iodide Symporter Metastable Intermediates Provide Insights into Conformational Transition between Principal Thermodynamic States

“…11 All-atom MD simulation studies of human NIS have been previously conducted to investigate the free energy of iodide binding 24 and to identify a putative ion translocation and gating pathway based on an inward-facing model. 25 A combination of structural data and MD simulations 26−28 have also elucidated most of the details of the transport mechanism of Aquifex aeolicus Na + -dependent leucine transporter (LeuT), 29 which shares the same fold as vSGLT. However, atomistic details pertaining to the conformational transition of NIS between its inwardly and outwardly open thermodynamic states in the presence and absence of bound ions, and the intermediate microstates that comprise this transition, have remained unclear.…”

“…All-atom MD simulation studies of human NIS have been previously conducted to investigate the free energy of iodide binding and to identify a putative ion translocation and gating pathway based on an inward-facing model . A combination of structural data and MD simulations − have also elucidated most of the details of the transport mechanism of Aquifex aeolicus Na + -dependent leucine transporter (LeuT), which shares the same fold as vSGLT.…”