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: 10436880
• 负责人: Mario A. Bianchet
• 金额: $ 45万
• 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/10436880
• 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; 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-转运体(NIS)是重要的质膜蛋白，介导主动的I-转运进入细胞内 甲状腺，甲状腺激素生物合成的第一步。NIS偶联i-与其电子的内向移位- 热化学梯度使Na+的内向运输顺着其电化学梯度下降。NIS的活性是产生电的， 用2Na+：1I-化学计量比。我们已经证明，NIS还运输环境污染物高氯酸盐 (ClO4-，一种I-转运抑制剂)，但电子中和(1NA+：1ClO4-)。NIS如何将不同的亚细胞 不同化学计量比的战略？我们在NIS中发现了一个高亲和力的非转运氧阴离子结合部位 当被ClO4-占据时，变构阻止两个Na+离子中的一个结合，改变化学结构- 从2Na+：1I-到1Na+：1I-的I-转运计量学。这降低了i-转运的驱动力，显著地降低了 折痕I-摄取。因此，受ClO4-污染的饮用水对人体健康的危害比污染前更大。 显然是这么想的。关于国家情报机构的一个关键问题是：在[I-]极低的情况下，国家情报机构如何有效地运输I-，S 在细胞外的环境中？我们在解决这个难题方面取得了重大进展。然而，完全 阐明NIS的传输机制将需要大量的进一步计算和实验。 在这些研究中，我们基于两个具有相同折叠的蛋白质的结构建立了NIS同源模型： VSGLT和SIAT。使用这些模型的MD模拟已经准确地预测了哪些残基起着关键作用 在NIS函数中，所有的预测都得到了实验证实。NIS的运输周期涉及16个物种： 1个空NiS，3个含1个离子的NiS物种，3个含2个离子的NiS物种，1个含3个离子的NiS物种 (8个态)和向内开放构象(8个态)。这16个物种可以认为是非常接近的 均衡。因此，可以通过测定布居数和自由能来描述NIS机制 在这16个物种中。我们建议使用MD模拟来确定组成ClO4-变构位置的残基 对于WT NIS和突变的NIS蛋白，我们已经证明了氧离子的运输是电生的，而不是电子中和的。 将对模拟所建议的构成变构位置的残基进行实验研究。我们 将通过计算搜索可能与变构位点结合的内源化合物，并通过实验 测试它们对NIS活动的影响。在我们的模拟中，在确定了可能与I-相互作用的NIS残基后，我们 将对它们进行实验研究。我们将使用定点突变、转运试验和动力学 全细胞和由纯化的NIS重组的蛋白脂质体中，电生理实验，闪烁 接近性分析、等温滴定量热法、统计热力学模型和计算方法。 ODS(MD模拟，包括元动力学)，以回答以下问题(具体目标)：1.什么是 NIS运输的总体机制？也就是说，所有物种的种群数量和自由能是多少 参与运输循环的？2.NIS识别I-是疏水阴离子吗？3.残基是什么 NIS中的非转运氧阴离子变构位置向上？