Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, AB T2N 1N4, Canada.
Imanis Life Sciences, Rochester, Minnesota 55901, United States.
J Chem Inf Model. 2020 Mar 23;60(3):1652-1665. doi: 10.1021/acs.jcim.9b01114. Epub 2020 Mar 12.
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) I uptake of 35 hNIS variants.
人源钠碘同向转运体（hNIS）是一种治疗诊断报告基因，可浓集多种已批准的 SPECT 和 PET 放射性示踪剂，作为甲状腺中的碘转运体在甲状腺激素合成中发挥重要作用。目前正在开发能够增强所需成像离子易位的 hNIS 突变体。不幸的是，由于缺乏对 hNIS 的 3D 结构及其与阴离子转运的关系的了解，这一工作受到了阻碍。hNIS 不存在任何构象状态的已知晶体结构。在这种情况下，同源建模可能非常有效；然而，由于 hNIS 与具有可用实验结构的相关二级转运体之间的序列同一性较低，因此选择模板和生成 3D 模型并非微不足道。在这里，我们报告了同源建模和 hNIS 半封闭状态下的分子动力学细化的联合应用。建模基于 LeuT 折叠蛋白家族的模板，并着重于对底物 - 离子结合口袋的细化。在这项工作中开发的共识模型与许多不同的 LeuT 折叠蛋白的现有生物物理和生化实验数据进行了比较。确定了一些功能上重要的残基，这些残基有助于形成共转运的 Na 离子和 I 底物的假定结合位点和渗透途径。通过生成 hNIS 的突变版本并测量 35 种 hNIS 变体的相对（相对于 WT hNIS）I 摄取，对模型预测进行了实验测试。
