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

 描述（由申请方提供）：Na+/I-同向转运体（NIS）是介导甲状腺和其他组织中I-转运的关键质膜蛋白。在甲状腺中，I-转运是甲状腺激素T3和T4生物合成的第一步。NIS将I-逆其电化学梯度的向内易位与Na+沿其电化学梯度的向内转运偶联。NIS活性是产电的，具有2 Na+：1 I-化学计量。相比之下，我们已经表明，NIS运输过氯酸盐（ReO 4-）和环境污染物高氯酸盐（ClO 4-）电中性。65年来，NIS介导的放射治疗一直是最成功的靶向内部放射癌症治疗，如甲状腺切除术后的甲状腺癌。自从我们克隆了NIS以来，NIS已经通过基因转移在甲状腺外癌中异位表达，使它们对放射性碘治疗敏感。NIS的研究具有相当大的基础和医学意义。通过研究患者中发现的先天性I-转运缺陷引起的NIS突变，我们对NIS的结构/功能关系有了重要的了解：特别是，我们正确地预测NIS将与细菌亮氨酸转运蛋白LeuT及其家族中的其他蛋白质具有相同的折叠。这种相似性的认识，使我们能够建立一个NIS同源性模型的基础上，另一种细菌蛋白的结构相同的折叠，vSGLT。分子动力学（MD）模拟使用我们的同源性模型准确地预测了哪些残基在NIS功能，底物特异性，动力学和化学计量中起着至关重要的作用。NIS在分子水平上的所有这些特征使我们能够提出NIS的I-转运机制。在这个项目中，我们将测试我们关于传输机制的假设。我们将为这项任务 一系列生物物理学方法，将基于全细胞的生化实验与使用纯化NIS [闪烁邻近测定法（SPA）和等温滴定量热法（ITC）]和计算分析（包括统计热力学（ST）和MD模拟）的研究相结合。我们还将测试具体的假设在运输周期中的个别残留物的作用。我们已经在解决NIS如何有效积累I的基本问题方面取得了相当大的进展-考虑到细胞外液中极低（亚μM）的I-浓度，这比蛋白质对卤化物的预期Kd低得多：我们已经确定第一个离子（Na+或I-）的结合将NIS对其他离子的亲和力增加了约10倍。将追求以下至关重要的具体目标：1。在存在不同浓度的其他离子的情况下，纯化的NIS对每种被运输的离子的亲和力是多少？结合到一个位点对其他位点的离子亲和力有什么影响？2.在向内开放的构象中，NIS对i所输送的离子的亲和力是多少？3.哪些残基在Na 2位点与Na+配位和/或参与将Na+释放到胞质溶胶中的途径？4.哪些残基与Na+在Na 1位点配位，它们如何决定单个氨基酸取代所带来的Na+/ReO 4-化学计量的变化？

 DESCRIPTION (provided by applicant): The Na+/I- symporter (NIS) is the key plasma membrane protein that mediates I- transport in the thyroid and other tissues. In the thyroid, I- transport is the first step in the biosynthesis of the thyroid hormones T3 and T4. NIS couples the inward translocation of I- against its electrochemical gradient to the inward transport of Na+ down its electrochemical gradient. NIS activity is electrogenic, with a 2 Na+:1 I- stoichiometry. By contrast, we have shown that NIS transports perrhenate (ReO4-) and the environmental pollutant perchlorate (ClO4-) electroneutrally. For over 65 years, NIS-mediated radiotherapy has been the most successful targeted internal radiation cancer treatment available, as administered in thyroid cancer post-thyroidectomy. Since we cloned it, NIS has been ectopically expressed by gene transfer in extra-thyroidal cancers, rendering them susceptible to radioiodide treatment. The study of NIS is of considerable basic and medical interest. We have gained significant insights into structure/function relations in NIS by studying congenital I- transport defect-causing NIS mutations found in patients: in particular, we correctly predicted that NIS would have the same fold as the bacterial leucine transporter LeuT and the other proteins in its family. The recognition of this similarity enabled us to build a NIS homology model based on the structure of another bacterial protein with the same fold, vSGLT. Molecular dynamics (MD) simulations using our homology model have accurately predicted which residues play crucial roles in NIS function, substrate specificity, kinetics, and stoichiometry. All this characterizatio of NIS at the molecular level has allowed us to propose a mechanism for I- transport by NIS. In this project, we will test our hypothesis about the transport mechanism. We will bring to this task a battery of biophysical approaches combining whole cell-based biochemical experiments with studies using purified NIS [scintillation proximity assays (SPA) and isothermal titration calorimetry (ITC)] and computational analysis, including statistical thermodynamics (ST) and MD simulations. We will also test specific hypotheses about the role of individual residues in the transport cycle. We have made considerable progress in addressing the fundamental question of how NIS can efficiently accumulate I- given the extremely low (sub-µM) I- concentrations in the extracellular fluids, which are much lower than the expected Kd of a protein for a halide: we have determined that binding of the first ion (either Na+ or I-) increases the affinity of NIS for he other ion by a factor of ~10. The following crucially important Specific Aims will be pursued: 1. What is the affinity of purified NIS for each of the transported ions in the presence of varying concentrations of the other ions? What is the effect of binding to one site on the ion affinities o the other sites? 2. What are the affinities of NIS in the inwardly open conformation for the ions i transports? 3. Which residues coordinate Na+ at the Na2 site and/or participate in the pathway that releases Na+ into the cytosol? 4. Which residues coordinate Na+ at the Na1 site, and how do they determine the change in Na+/ReO4- stoichiometry brought about by single amino acid substitutions?