Molecular mechanisms of bacterial homologs of neurotransmitter:sodium symporters

6.项目摘要/摘要 神经递质：钠转运体(NSS)将底物的积累与钠的运动结合起来 离子向下穿过质膜的浓度梯度，因此构成了 细胞信号和动态平衡。神经递质包括多巴胺、5-羟色胺和 去甲肾上腺素-苯丙胺、可卡因和抗抑郁药物的靶标-以及 GABA和甘氨酸，用于治疗癫痫和精神分裂症。2005年，Gouaux集团 Leut是一种细菌NSS同系物，在1.65℃下溶解，与1个Leu和2个Na+结合在一个 闭塞结合口袋(称为初级底物结合(S1)部位)。这种结构并不容易 提示底物从胞外或胞内侧到S1位点的路径。意想不到的 第二底物结合位点(S2)位于细胞外前庭 结合和助熔剂实验表明，两个结合部位可以同时占据。 S2位的底物变构地触发细胞内Na+和来自S1位的底物的释放， 从而起到“符号效应器”的作用。因为三环抗抑郁剂(TCA)与该S2的结合方式不同 它们不会促进底物从S1位点释放，从而起到联合解偶联剂的作用 运输。确定与运输和渗透途径相关的构象变化 是该项目的长期目标，对理解功能至关重要 人类神经递质转运体的机制以及药物如何作用于这些机制。至 为了实现这一目标，已经开发了基于与 在计算建模、膜蛋白结晶学方面有专长的研究人员(哈雷尔·韦恩斯坦) (保尔·尼森)和单分子荧光光谱(斯科特·布兰查德)使组合 本申请中描述的多学科方法。提出了以下具体目标：1)使用 我们关于S2结合的特异性和调节的新发现，通过洗涤剂、突变和 离子取代，以开发条件，使我们能够了解调节Leut的性质 并解决了底物与S2结合位点结合的Leut结构。这将提供原子 解析数据为我们关于底物结合到的运输中的重要作用的机械性假说提供信息 这个网站。2)根据特定的构象变化来描述底物运输的机制 在将底物结合到S2-位点所触发的变构信号传播到 细胞内门的转运蛋白，并允许向内释放底物。3)建立我们的 细菌转运蛋白的结构和功能研究有助于了解SERT和DAT的功能。我们 将：a)证明S2位点在这些人类转运蛋白中的基本功能作用，以及b)使用一个氯-- 依赖Leut突变体来确定氯离子结合部位的结构，从而解释其功能 氯离子在SERT和DAT中的作用

6. Project Summary/Abstract Neurotransmitter:sodium symporters (NSS) couple the accumulation of substrate to the movement of sodium ions down their concentration gradient across the plasma membrane, and as such constitute key elements in cellular signaling and homeostasis. NSS include the transporters for dopamine, serotonin and norepinephrine-targets for amphetamine, cocaine, and antidepressant drugs-as well as the transporters for GABA and glycine, which are targeted for treatment of epilepsy and schizophrenia. In 2005 the Gouaux group solved at 1.65 ¿ the structure of LeuT, a bacterial NSS homolog, crystallized with 1 Leu and 2 Na+ bound in an occluded binding pocket (referred to as primary substrate binding (S1) site). The structure provided no easy clues to the pathway of substrate to the S1 site from the extracellular or the intracellular side. An unexpected second substrate binding (S2) site located in the extracellular vestibule was identified during the previous project period; binding and flux experiments showed that the two binding sites can be occupied simultaneously. Substrate in the S2 site allosterically triggers intracellular release of Na+ and substrate from the S1 site, thereby functioning as a "symport effector." Because tricyclic antidepressants (TCA) bind differently to this S2 site, they do not promote substrate release from the S1 site and thus act as symport uncouplers to inhibit transport. Identifying the conformational changes associated with transport and the permeation pathways that are formed within the transporter are long term goals of this project critical to understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. To achieve this goal, an integrated approach has been developed based on active collaborations with investigators whose expertise in computational modeling (Harel Weinstein), membrane protein crystallography (Poul Nissen), and single-molecule fluorescence spectroscopy (Scott Blanchard) enables the combined multidisciplinary approach described in this application. The following specific aims are proposed: 1) To use our novel discoveries regarding the specificity and modulation of S2 binding, by detergents, mutations, and ionic substitution, to develop conditions that enable us to understand the regulation of LeuT properties by the S2 binding site and to solve a structure of LeuT with substrate bound to the S2 site. This will provide atomic resolution data to inform our mechanistic hypothesis as to the essential role in transport of substrate binding to this site. 2) To characterize the mechanism of substrate transport in terms of specific conformational changes in the transporter that propagate the allosteric signal triggered by substrate binding to the S2-site towards the intracellular gate of the transporter and allow inward release of substrate. 3) To establish the relevance of our structural and functional findings in bacterial transporters to understanding the function of SERT and DAT. We will: a) demonstrate the essential functional role of the S2 site in these human transporters, and b) use a Cl-- dependent LeuT mutant to determine the structure of the Cl- binding site and thus to explicate the functional role of Cl- in SERT and DAT.