Computational studies of membrane transport proteins

膜转运蛋白的计算研究

• 批准号: 9358608
• 负责人: Lucy Forrest
• 金额: $ 83.58万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9358608
• 关键词: ATP Hydrolysis; Address; Affinity; Amino Acid Transporter; Amino Acids; Antidepressive Agents; Architecture; Aspartate; Back; Bacterial Proteins; Binding; Binding Sites; Biochemical; Brain; Carrier Proteins; Cellular Membrane; Collaborations; Coupled; Data; Diabetes Mellitus; Docking; Elevator; Employee Strikes; Energy-Generating Resources; Excitatory Amino Acids; Family; Fluorometry; Glutamate Transporter; Glutamates; Homologous Gene; Homology Modeling; Human; Integral Membrane Protein; Ions; Journals; Kidney; Knowledge; Laboratories; Light; Location; Measurement; Membrane; Membrane Proteins; Membrane Transport Proteins; Mental Depression; Methods; Modeling; Modification; Molecular; Molecular Conformation; Motion; Movement; Mutate; National Institute of Neurological Disorders and Stroke; Nature; Neurons; Neurotransmitters; Nutrient; Organism; Orthologous Gene; Paroxetine; Pathway interactions; Pharmaceutical Preparations; Pharmacological Treatment; Physiological; Protein Conformation; Proteins; Recycling; Reporting; Resolution; Role; Shapes; Side; Site; Sodium; Specificity; Structural Models; Structure; Succinates; Testing; Universities; aqueous; base; biophysical techniques; computer studies; design; dicarboxylate-binding protein; extracellular; feeding; improved; inhibitor/antagonist; inorganic phosphate; insight; interest; molecular dynamics; neurotransmitter uptake; novel; novel therapeutics; presynaptic neurons; research study; response; serotonin transporter; small molecule; sodium ion; symporter; three dimensional structure; uptake; voltage clamp

Secondary active transporters are a class of membrane proteins that utilize pre-existing molecular concentration gradients as an energy source for translocating another substrate, such as a nutrient or a neurotransmitter, against its concentration gradient. They do so by changing conformations so as to form a pathway to the substrate binding site(s) on one or other side of the membrane, in a cycle known as alternating access. Every organism expresses dozens of different secondary transporter proteins, based on a diverse set of different architectures, albeit always with some form of internal structural symmetry. In spite of the unprecedented insights from the recently reported three-dimensional structures, a detailed understanding of the mechanism of each membrane transport protein requires knowledge of its structure in many more conformational states, as well as identification of the binding regions for the substrate or substrates. Studies from our group over the last year have provided such insights into a number of biomedically important transporters responsible for inorganic phosphate, succinate or neurotransmitter uptake, as detailed below. In 2014, we predicted the structural fold of a secondary transporter responsible for sodium-coupled phosphate uptake in the kidney, called NaPi-IIa, by identifying an evolutionary relationship with a a sodium-coupled dicarboxylate transporter known as VcINDY, whose structure was then used as a template for homology modeling. Within that model we had proposed binding sites for the substrates (Fenollar-Ferrer et al, Biophysical Journal, 2014; Fenollar-Ferrer et al, Biophysical J, 2015), but the protein had a rather unusual structure, with a large aqueous extracellular cavity, and the other states required for alternating access were enigmatic. We therefore used our previously-developed repeat-swap modeling approach to predict an alternate conformational state for the template protein, VcINDY. The resultant structural model strongly indicated a two-domain elevator-type conformational mechanism, similar to that previously described for the glutamate transporter family (Reyes et al, Nature 2008; Crisman et al, Proc Natl Acad Sci 2009), a mechanism with significant implications for its interaction with the membrane. To test this striking prediction, our collaborators in the Mindell laboratory here at NINDS, used biochemical and biophysical approaches to probe the predicted state (1). The results of these experiments provided strong support that the elevator-like mechanism of secondary transporters is more common than previously anticipated. We subsequently used the new repeat-swapped model of VcINDY as a template for a new model of NaPi-II, which as expected, also predicts an elevator-like motion, and moreover could be used to aid interpretation of elegant voltage-clamp fluorometry measurements performed by the Forster laboratory (2). Both studies were highlighted in their respective journals as notable contributions (Ryan and Vandenberg, Nature Struct Mol Biol, 2016; Gasnier, Biophys J, 2016). Aside from the identification of the conformational states required for alternating access, a major unresolved question for many secondary active transporters is how they bind and respond to their substrates and, by contrast, how inhibitors interfere with their mechanisms. In particular, a long-standing interest of our laboratory is the manner by which neurotransmitters are recycled into the presynaptic neuron by sodium-driven transporters in the neurotransmitter:sodium symporter (NSS) and excitatory amino acid transporter (EAAT) families. In fact, the details of substrate and inhibitor interactions remain poorly understood in many of those transporters. Even one of the highest affinity NSS inhibitors, the antidepressant paroxetine, has a binding mode that has remained ambiguous even in the light of recent high-resolution crystal structures of its target, serotonin transporter (SERT; Coleman et al, Nature, 2016). We used structure prediction and docking methods to predict the binding of paroxetine to SERT orthologs from three different species (3). Experimental modification of the predicted interactions the group of Satinder Singh at Yale University, revealed components of the binding site that are critical for paroxetine specificity. Our study therefore provides an important step towards the rational design of novel and improved antidepressants. Transport and inhibition of NSS transporters requires binding of sodium ions, which are thought to control the conformation of the protein by an unknown mechanism. In a separate study, in collaboration with the Rudnick lab at Yale University, we provided insights into the sodium effect on conformation in an NSS homolog called LeuT, by molecular dynamics simulations of the protein and of mutated versions thereof (4). Together our results indicate that just one of the two sodium binding sites is critical for trapping the transporter into an inhibitor/substrate-binding orientation, whereas a second sodium has more indirect and smaller effects on the conformation of the protein. Finally, we also helped provide a molecular description of the interaction between EAATs and their substrate, by building a homology model of a human transporter based on the structure of a bacterial homolog. We identified amino acids that differ between the human and bacterial proteins, underlying differences in specificity for glutamate and aspartate (5). The Kanner (Jerusalem) and Fahlke (Hannover) labs tested these hypotheses, and identified a single amino acid group as critical for glutamate specificity in neuronal excitatory uptake. The aforementioned studies contribute to furthering our understanding of neurotransmitter recycling in the brain.

次级活性转运蛋白是一类膜蛋白，它利用预先存在的分子浓度梯度作为能量源来转运另一种底物，如营养物质或神经递质，以对抗其浓度梯度。它们通过改变构象来形成一条通往膜一侧或另一侧的底物结合部位(S)的路径，这一循环被称为交替访问。每个生物体都表达数十种不同的次级转运蛋白，基于一组不同的不同结构，尽管它们总是具有某种形式的内部结构对称性。尽管最近报道的三维结构具有前所未有的洞察力，但要详细了解每种膜运输蛋白的机制，需要了解其在更多构象状态下的结构，以及识别底物或底物的结合区。我们小组过去一年的研究为许多生物医学上重要的转运蛋白提供了这样的见解，这些转运蛋白与无机磷、琥珀酸或神经递质的摄取有关，如下所述。 2014年，我们预测了负责肾脏摄取钠偶联磷酸盐的二级转运蛋白Napi-IIa的结构折叠，方法是确定与名为VcINDY的钠偶联二羧酸盐转运蛋白的进化关系，然后将其结构用作同源建模的模板。在该模型中，我们提出了底物的结合位点(Fenollar-Ferrer等人，生物物理杂志，2014；Fenollar-Ferrer等人，生物物理杂志，2015)，但蛋白质具有相当不寻常的结构，具有巨大的水胞外腔，而交替访问所需的其他状态是谜。因此，我们使用我们之前开发的重复交换建模方法来预测模板蛋白VcINDY的替代构象状态。所得到的结构模型强烈表明了一种双域升降型构象机制，类似于先前对谷氨酸转运蛋白家族的描述(Reyes等人，自然2008；Crisman等人，Proc Natl Acad Sci 2009)，这一机制对其与膜的相互作用具有重要意义。为了测试这一惊人的预测，我们在NINDS明德尔实验室的合作者使用生化和生物物理方法来探测预测的状态(1)。这些实验结果有力地支持了次级转运体的类电梯机制比先前预期的更常见。随后，我们使用VcINDY的新的重复交换模型作为新模型的NAPI-II的模板，正如预期的那样，它也预测了电梯般的运动，而且可以用来帮助解释Forster实验室进行的优雅的电压钳荧光测量(2)。这两项研究在各自的期刊上都被强调为值得注意的贡献(Ryan和Vandenberg，自然结构Mol Biol，2016；Gasnier，生物物理学J，2016)。 除了识别交替进入所需的构象状态外，许多次级活性转运体的一个主要悬而未决的问题是它们如何与底物结合并做出反应，以及相反，抑制剂如何干扰它们的机制。特别是，我们实验室长期以来一直感兴趣的是神经递质如何通过神经递质中的钠驱动转运体：钠转运体(NSS)和兴奋性氨基酸转运体(EAAT)家族循环进入突触前神经元。事实上，底物和抑制物相互作用的细节在许多转运蛋白中仍然知之甚少。即使是亲和力最高的NSS抑制剂之一，抗抑郁剂帕罗西汀，也有一种结合模式仍然不明确，即使根据其目标5-羟色胺转运体最近的高分辨率晶体结构(SERT；Coleman等人，《自然》，2016)。我们使用结构预测和对接方法预测了帕罗西汀与来自三个不同物种的SERT同源物的结合(3)。耶鲁大学的Satinder Singh小组对预测的相互作用进行了实验修正，揭示了结合部位的成分对帕罗西汀的特异性至关重要。因此，我们的研究为合理设计新型和改进的抗抑郁药物提供了重要的一步。NSS转运体的运输和抑制需要钠离子的结合，钠离子被认为通过一种未知的机制控制蛋白质的构象。在与耶鲁大学鲁德尼克实验室合作的另一项研究中，我们通过对蛋白质及其突变版本的分子动力学模拟，深入了解了钠对名为Leut的NSS同系物构象的影响。总之，我们的结果表明，两个钠结合位点中的一个对于将转运蛋白捕获到抑制物/底物结合方向是关键的，而第二个钠对蛋白质的构象有更多和更小的间接影响。最后，我们还通过建立基于细菌同源物结构的人类转运蛋白的同源模型，帮助提供了EAAT与其底物之间相互作用的分子描述。我们确定了人类和细菌蛋白质之间的不同氨基酸，即谷氨酸和天冬氨酸的潜在特异性差异(5)。Kanner(耶路撒冷)和Fahlke(汉诺威)实验室测试了这些假说，并确定单一氨基酸基团对神经元兴奋性摄取中谷氨酸专一性至关重要。上述研究有助于加深我们对大脑中神经递质循环的理解。