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: 10580384
• 负责人: Mario A. Bianchet
• 金额: $ 7.47万
• 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/10580384
• 关键词: Affinity; Allosteric Site; Anions; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Calorimetry; Cell membrane; Cells; Characteristics; Computer Analysis; Computing Methodologies; Couples; Electrophysiology (science); Environmental Pollutants; Equilibrium; Free Energy; Goals; Health; Homology Modeling; Human; Hydrophobicity; Ion Transport; Ions; Kinetics; Mediating; Membrane; Membrane Proteins; Modeling; Molecular Conformation; Perchlorates; Play; Population; Production; Proteins; Research; SLC5A5 gene; Site-Directed Mutagenesis; Sodium Iodide; Structure; Testing; Thermodynamics; Thyroid Gland; Thyroid Hormones; Titrations; Uncertainty; base; contaminated drinking water; driving force; environmental transport; experimental study; extracellular; hormone biosynthesis; inhibitor; innovation; mutant; 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- 同向转运蛋白 (NIS) 是介导活性 I- 转运至细胞的关键质膜蛋白。 甲状腺，甲状腺激素生物合成的第一步。 NIS 将 I- 的向内易位与其电耦合 Na+沿着其电化学梯度向内运输的化学梯度。 NIS 活性是生电的， 具有 2Na+:1I- 化学计量。我们已经证明 NIS 还传输环境污染物高氯酸盐 （ClO4-，I- 转运抑制剂），但呈电中性（1Na+:1ClO4-）。 NIS如何转位不同的子 具有不同化学计量的策略？我们在 NIS 中发现了一个高亲和力非转运氧阴离子结合位点 当被 ClO4- 占据时，变构地阻止两个 Na+ 离子之一结合，改变化学性质 从 2Na+:1I- 到 1Na+:1I- 的 I- 运输的化学计量。这降低了 I- 传输的驱动力，显着降低了 增加 I- 吸收。因此，被ClO4-污染的饮用水对人体健康的危害比以前更严重。 狠狠地想。关于 NIS 的一个关键问题是：考虑到极低的 [I-]s，NIS 如何有效地传输 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。尽管如此，充分 阐明 NIS 的传输机制将需要大量的进一步计算和实验。 对于这些研究，我们基于具有相同折叠的两种蛋白质的结构构建了 NIS 同源模型： vSGLT 和 SiaT。使用这些模型的 MD 模拟准确预测了哪些残基发挥关键作用 在NIS功能中；所有的预测都已被实验证实。 NIS传输循环涉及16种： 1 个空 NIS、3 个具有一种离子的 NIS、3 个具有两个离子的 NIS 和 1 个具有三个离子的 NIS （8 个状态）和向内开放构象（8 个状态）。这16个物种可以认为是非常接近的 平衡。因此，NIS机制可以通过确定粒子数和自由能来描述 16个物种中。我们建议使用 MD 模拟来识别构成 ClO4-变构位点的残基 我们已经证明，WT NIS 和突变型 NIS 蛋白可以通过电而不是电中性方式运输氧阴离子。 模拟建议的组成变构位点的残基将通过实验进行研究。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。在我们的模拟中确定了可能与 I- 相互作用的 NIS 残基后，我们 将通过实验研究它们。我们将使用定点诱变、转运分析和动力学 全细胞和用纯化的 NIS 重建的脂蛋白体、电生理实验、闪烁 邻近分析、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）来回答以下问题（具体目标）： 1. 什么是 NIS传输的整体机制？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？ 2. NIS 是否将 I- 识别为疏水性阴离子？ 3. 残留物会产生什么 NIS 中的非运输氧阴离子变构位点？