Molecular mechanisms of bacterial homologs of neurotransmitter:sodium symporters

描述（由申请人提供）：神经递质：钠同调蛋白（NSS）将底物的积累与钠离子沿其浓度梯度穿过质膜的运动结合起来，因此构成细胞信号传导和体内平衡的关键要素。NSS包括多巴胺、血清素和去甲肾上腺素的转运蛋白（安非他明、可卡因和抗抑郁药物的目标），以及GABA和甘氨酸的转运蛋白（用于治疗癫痫和精神分裂症）。2005年，Gouaux小组以1.65°c解算了细菌NSS同源物LeuT的结构，该结构在一个封闭的结合口袋（称为初级底物结合（S1）位点）中结晶为1 Leu和2 Na+。这种结构无法提供底物从细胞外或细胞内到达S1位点的简单线索。在上一个项目期间，发现了位于细胞外前庭的意想不到的第二底物结合（S2）位点；结合和通量实验表明，这两个结合位点可以同时被占用。S2位点的底物变构性地触发细胞内Na+和S1位点底物的释放，从而发挥“对称效应”的作用。由于三环抗抑郁药（TCA）与S2位点的结合方式不同，它们不能促进底物从S1位点释放，因此可以作为同义转运解偶联剂来抑制转运。确定与转运相关的构象变化和转运体内部形成的渗透途径是本项目的长期目标，对于理解人类神经递质转运体的功能机制以及药物如何作用于这些机制至关重要。为了实现这一目标，基于与研究人员的积极合作，开发了一种综合方法，这些研究人员在计算建模（Harel Weinstein），膜蛋白晶体学（Poul Nissen）和单分子荧光光谱（Scott Blanchard）方面的专业知识使本应用程序中描述的多学科方法相结合。我们提出了以下具体目标：1)利用我们关于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，创造条件，使我们能够理解S2结合位点对LeuT性质的调节，并解决底物与S2位点结合的LeuT结构。这将提供原子分辨率数据，以告知我们的机制假设，即底物结合转运到该位点的重要作用。2)从转运体的特定构象变化来表征底物运输的机制，这些构象变化将底物与s2位点结合引发的变构信号传播到转运体的胞内门，并允许底物向内释放。3)建立我们在细菌转运体中的结构和功能发现的相关性，以了解SERT和DAT的功能。我们将：a)证明S2位点在这些人类转运体中的重要功能作用，b)使用Cl-依赖的LeuT突变体来确定Cl-结合位点的结构，从而阐明Cl-在SERT和DAT中的功能作用。公共卫生相关性：神经递质转运体是可卡因和安非他明等精神兴奋剂药物的靶标，也是抗抑郁药以及正在开发的治疗癫痫和精神分裂症的新药的靶标。确定转运的构象动力学、转运体内部的渗透途径以及底物和抑制剂结合的作用对于理解人类神经递质转运体的功能机制以及药物如何作用于这些机制至关重要。我们已经建立的强有力的方法将使我们能够达到这种理解，并解决药物作用和治疗设计指南，这些指南以坚实的结构和功能信息为基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: Neurotransmitter transporters are the target of psychostimulant drugs such as cocaine and amphetamine and are targets for antidepressants as well as for new drugs in development for the treatment of epilepsy and schizophrenia. Identifying the conformational dynamics of transport, the permeation pathways within the transporter, and the role of substrate and inhibitor binding are critical for understanding the functional mechanisms of the human neurotransmitter transporters and how drugs act upon these mechanisms. The powerful approaches we have established will allow us to reach this understanding and address drug action and guidelines for therapy design anchored in solid structural and functional information.