Mechanism of I- transport by the Na+/l- symporter (NIS)

Na /l- 同向转运体 (NIS) 的 I- 转运机制

• 批准号: 10219677
• 负责人: Mario A. Bianchet
• 金额: $ 47.22万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10219677
• 关键词: Affinity; Allosteric Site; Amino Acid Substitution; Anions; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Birth; Calorimetry; Cell membrane; Cells; Characteristics; Cloning; Computer Analysis; Computing Methodologies; Couples; Data; Defect; Development; Electrophysiology (science); Environmental Pollutants; Equilibrium; Free Energy; Gene Transfer; Goals; Health; Homology Modeling; Human; Hydrophobicity; Impaired cognition; Individual; Ion Transport; Ions; Kinetics; Malignant neoplasm of thyroid; Measurement; Mediating; Membrane; Membrane Potentials; Membrane Proteins; Modeling; Molecular Conformation; Mutation; Nature; Oncogenes; Perchlorates; Physiological; Play; Population; Production; Property; Proteins; Public Health; Regulation; Research; SLC5A5 gene; Site; Site-Directed Mutagenesis; Sodium Iodide; Structure; Testing; Thermodynamics; Thyroid Gland; Thyroid Hormones; Thyroidectomy; Time; Titrations; Uncertainty; base; cancer therapy; contaminated drinking water; drinking water; driving force; environmental transport; experimental study; extracellular; hormone biosynthesis; inhibitor/antagonist; innovation; internal radiation; mutant; novel; prevent; protein transport; proteoliposomes; reconstitution; simulation; stoichiometry; symporter; uptake

The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates active I- transport into the thyroid, the first step in thyroid hormone biosynthesis. NIS couples the inward translocation of I- against its elec- trochemical gradient to the inward transport of Na+ down its electrochemical gradient. NIS activity is electrogenic, with a 2Na+:1I- stoichiometry. We have shown that NIS also transports the environmental pollutant perchlorate (ClO4-, an inhibitor of I- transport), but electroneutrally (1Na+:1ClO4-). How does NIS translocate different sub- strates with different stoichiometries? We discovered a high-affinity non-transport oxyanion binding site in NIS that, when occupied by ClO4-, allosterically prevents one of the two Na+ ions from binding, changing the stoi- chiometry of I- transport from 2Na+:1I- to 1Na+:1I-. This reduces the driving force for I- transport, markedly de- creasing I- uptake. Thus, drinking water contaminated with ClO4- is more deleterious to human health than pre- viously thought. A crucial question about NIS is: how can NIS efficiently transport I-, given the extremely low [I-]s in the extracellular milieu? We have made significant progress toward solving this puzzle. Nevertheless, fully elucidating the mechanism of transport by NIS will require extensive further computation and experimentation. For these studies, we built NIS homology models based on the structures of two proteins with the same fold: vSGLT and SiaT. MD simulations using these models have accurately predicted which residues play key roles in NIS function; all predictions have been experimentally confirmed. The NIS transport cycle involves 16 species: 1 empty NIS, 3 NIS species with one ion, 3 NIS species with two ions, and 1 NIS with three ions, in the outwardly (8 states) and the inwardly open conformation (8 states). The 16 species can be considered to be very close to equilibrium. Therefore, the NIS mechanism can be described by determining the populations and free energies of the 16 species. We propose to identify the residues making up the ClO4- allosteric site using MD simulations of WT NIS and mutant NIS proteins we have shown to transport oxyanions electrogenically, not electroneutrally. The residues suggested by the simulations to make up the allosteric site will be investigated experimentally. We will search computationally for endogenous compounds that may bind to the allosteric site, and experimentally test their effects on NIS activity. Having identified NIS residues that likely interact with I- in our simulations, we will investigate them experimentally. We will use site-directed mutagenesis, transport assays and kinetics in whole cells and in proteoliposomes reconstituted with purified NIS, electrophysiological experiments, scintillation proximity assays, isothermal titration calorimetry, statistical thermodynamics modeling, and computational meth- ods (MD simulations, including metadynamics) to answer the following questions (Specific Aims): 1. What is the overall mechanism of NIS transport? That is, what are the populations and the free energies of all the species that participate in the transport cycle? 2. Does NIS recognize I- as a hydrophobic anion? 3. What residues make up the non-transport oxyanion allosteric site in NIS?

Na+/i-分别蛋白（NIS）是介导活性I-转运到该的关键质膜蛋白 甲状腺，甲状腺激素生物合成的第一步。 nis夫妻夫妇向内易位I-与其电子的易位 将Na+向下运输的梯度向下进行电化学梯度。 NIS活性是电源的， 具有2NA+：1-stoictimementry。我们已经表明，NIS还运输环境污染物高氯酸盐 （clo4-，i-转运的抑制剂），但电屈（1NA+：1CLO4-）。 NIS如何转移不同的子 - 带有不同的化学计量法？我们在NIS中发现了一个高亲和力的非传输氧结合位点 当被clo4-占据时，变构可防止两个Na+离子之一结合，改变了stoi- i-转运的数量法从2NA+：1-1-NA+：1-。这降低了I-运输的驱动力，显着地 creas绕。因此，被Clo4-污染的饮用水对人类健康更有害于人类健康 狡猾地想。关于NIS的一个关键问题是：鉴于非常低[I-] S，NIS如何有效地运输I- 在细胞外环境中？我们在解决这个难题方面取得了重大进展。但是，完全 通过NIS阐明运输机制将需要广泛的进一步计算和实验。 对于这些研究，我们基于两个具有相同折叠的蛋白质的结构建立了NIS同源性模型： VSGLT和SIAT。使用这些模型的MD模拟可以准确预测哪些残基扮演关键角色 在NIS功能中；所有预测均已实验证实。 NIS运输周期涉及16种： 1个空的NIS，3个具有一个离子的NIS种，3个离子的3个NIS物种，1个NIS，带有3个离子，在外部 （8个州）和内向开放的构象（8个州）。可以认为16种非常接近 平衡。因此，可以通过确定种群和自由能来描述NIS机制 在16种。我们建议使用MD模拟识别构成CLO4变构位点的残基 WT NIS和突变NIS蛋白的蛋白质已证明是通过电源而不是电源性转运的。 模拟建议构成变构位点的残基将进行实验研究。我们 将在计算上搜索可能与变构位点结合并实验的内源化合物 测试它们对NIS活动的影响。确定了可能与i-相互作用的NIS残留物，我们 将通过实验调查它们。我们将在地点定向诱变，运输分析和动力学中 全细胞和蛋白质脂质体中用纯化的NIS重组，电生理实验，闪烁 接近测定，等温滴定量热法，统计热力学建模和计算甲基 ODS（MD模拟，包括元动力学）回答以下问题（特定目的）：1。 NIS运输的总体机制？也就是说，所有物种的种群和自由能力是什么 参与运输周期？ 2。尼斯是否将i-识别为疏水阴离子？ 3。残留物有什么 在NIS上升高非传输氧构构位？