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: 10683934
• 负责人: Mario A. Bianchet
• 金额: $ 44.41万
• 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/10683934
• 关键词: Affinity; Allosteric Site; Amino Acid Substitution; Anions; Binding; Binding Sites; Biochemical; Biological; Biological Assay; Birth; Calorimetry; Cell membrane; Cells; Characteristics; Cloning; Collaborations; 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; Iodides; 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; Predisposition; Production; Property; Proteins; Public Health; Regulation; Research; Site; Site-Directed Mutagenesis; Sodium; Structure; Testing; Thermodynamics; Thyroid Gland; Thyroid Hormones; Thyroidectomy; Time; Titrations; Uncertainty; 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-同向转运体（NIS）是介导I-主动转运到细胞膜的关键膜蛋白， 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β- Na+沿其电化学梯度向内运输。NIS活动是产电的， 以2Na+：1 I-化学计量。我们已经表明，NIS也运输环境污染物高氯酸盐 （ClO 4-，I-转运的抑制剂），但电中性（1 Na+：1ClO 4-）。NIS是如何在不同的亚细胞中 不同化学计量的底物我们在NIS中发现了一个高亲和力的非转运氧阴离子结合位点 当被ClO 4-占据时，变构阻止两个Na+离子中的一个结合，改变了化学计量。 I-从2Na+：1 I-向1 Na+：1 I-转运的化学计量学。这降低了I-运输的驱动力，显著地降低了- 增加碘摄入量。因此，被ClO 4-污染的饮用水对人体健康的危害比未被污染的饮用水更大。 刘翔想。关于NIS的一个关键问题是：在[I-]极低的情况下，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。然而，充分 阐明NIS的传输机制将需要进一步的大量计算和实验。 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模拟准确地预测了哪些残基起关键作用 在NIS功能;所有的预测都得到了实验证实。NIS的运输循环涉及16个物种： 1个空NIS、3个具有一个离子的NIS物质、3个具有两个离子的NIS物质和1个具有三个离子的NIS，在向外的 （8个状态）和向内开放构象（8个状态）。这16个物种可以被认为是非常接近 均衡因此，可以通过确定粒子数和自由能来描述NIS机制 16个物种。我们建议使用分子动力学模拟来确定ClO 4-变构位点的残基 野生型NIS和突变型NIS蛋白质，我们已经证明，运输含氧阴离子产电，而不是电中性。 模拟建议的残基，以弥补变构网站将进行实验研究。我们 将通过计算搜索可能与变构位点结合的内源性化合物，并通过实验 测试它们对NIS活性的影响。在我们的模拟中，我们已经确定了可能与I-相互作用的NIS残基， 将对它们进行实验研究。我们将使用定点诱变，转运试验和动力学， 全细胞和用纯化NIS重构的蛋白脂质体，电生理学实验，闪烁 邻近分析、等温滴定量热法、统计热力学建模和计算方法， ods（MD模拟，包括元adaptics）回答以下问题（具体目标）：1.是什么 NIS传输的总体机制？也就是说，所有物种的种群和自由能是多少 参与运输循环的生物2. NIS是否将I-识别为疏水阴离子？3.残留物 增加NIS中的非转运氧阴离子变构位点？

项目成果

Structural insights into the mechanism of the sodium/iodide symporter.

• DOI: 10.1038/s41586-022-05530-2
• 发表时间: 2022-12
• 期刊: NATURE
• 影响因子: 64.8
• 作者: Ravera, Silvia;Nicola, Juan Pablo;Salazar-De Simone, Glicella;Sigworth, Fred J.;Karakas, Erkan;Amzel, L. Mario;Bianchet, Mario A.;Carrasco, Nancy
• 通讯作者: Carrasco, Nancy