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

 描述(由申请人提供)：Na+/I-转运体(NIS)是关键的质膜蛋白，介导甲状腺和其他组织中的I-转运。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将与其电化学梯度相反的I-向内转移与Na+沿其电化学梯度向内转移相耦合。NiS的活性是电生的，具有2Na+：1I-化学计量比。相比之下，我们已经表明，NIS以电子中和的方式运输高氯酸盐(ReO4-)和环境污染物高氯酸盐(ClO4-)。65年来，NIS介导的放射治疗一直是现有的最成功的靶向内放射癌症治疗方法，用于甲状腺切除术后的甲状腺癌。自从我们克隆它以来，NIS已经通过基因转移在甲状腺外癌症中异位表达，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价值。通过研究在患者中发现的先天性I-转运缺陷导致的NIS突变，我们对NIS的结构/功能关系有了重要的见解：特别是，我们正确地预测了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。对这种相似性的认识使我们能够基于另一种具有相同折叠的细菌蛋白vSGLT的结构建立NIS同源模型。使用我们的同源模型进行的分子动力学(MD)模拟已经准确地预测了哪些残基在NIS功能、底物专一性、动力学和化学计量学中发挥关键作用。所有这些在分子水平上对NIS的表征使我们能够提出一种NIS的I-转运机制。在这个项目中，我们将检验我们关于传输机制的假设。我们将为这项任务带来 一系列生物物理方法结合了基于全细胞的生化实验、使用纯化的NIS[闪烁邻近分析(SPA)和等温滴定热计(ITC)]的研究以及计算分析，包括统计热力学(ST)和MD模拟。我们还将测试有关单个残基在运输周期中的作用的具体假设。在解决NIS如何有效积累I-的根本问题上，我们已经取得了相当大的进展，因为胞外液中的I-浓度极低(亚微米)，远远低于蛋白质对卤化物的预期Kd：我们已经确定，第一个离子(Na+或I-)的结合使NIS与其他离子的亲和力增加了~10倍。我们将追求以下至关重要的具体目标：1.在不同浓度的其他离子存在的情况下，纯化的NIS对每个运输离子的亲和力是什么？与一个位点的结合对其他位点的离子亲和力有什么影响？2.离子I转运的内向开放构象中的NiS的亲和力是什么？3.哪些残基与Na2位上的Na+配位和/或参与Na+释放到细胞质中的途径？4.哪些残基与Na1位上的Na+配位，它们如何决定单一氨基酸取代带来的Na+/ReO4化学计量比的变化？

 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?