LeuT Membrane Transporter Dynamics Determined by EPR and Computational Techniques

描述(申请人提供)：5-羟色胺、多巴胺和去甲肾上腺素神经递质转运体(分别为SERT、DAT和NET)参与了从高血压、心力衰竭到帕金森氏症等一系列与生理和心理健康相关的过程。SERT和DAT也是可卡因和安非他明的结合靶标。阐明这些转运蛋白调节底物流动所经历的主要构象变化将有助于开发药物化合物来对抗这些疾病，因为它提供了对这些复杂的分子机器如何工作的更好的理解。SERT、DAT和NET属于神经递质钠转运体(NSS)转运蛋白家族。目前，NSS家族的任何成员都不存在高分辨率结构。然而，细菌同系物Leut的高分辨率晶体结构为这一重要的转运蛋白家族的结构和底物结合提供了见解。然而，尽管这种晶体结构突出了运输周期中的单一关闭-关闭状态，但对功能至关重要的多种其他构象仍然未知。目前的工作将利用NSS同源Leut晶体结构和电子顺磁共振(EPR)距离和可及性数据来研究底物通道是如何通过构象变化来控制的。为了实现这一目的，将开发一种计算算法，该算法将提供Leut从一个状态到另一个状态的连续图像。第二种状态将由围绕Leut门控残基收集的EPR数据来描述。该算法将包括一个能量最小化过程，该过程由EPR数据驱动，通过生物相关的途径达到第二构象。这种方法将描述Leut如何在结构上改变以控制运输。为Leut确定的结构动力学将为NSS家族的同源转运蛋白提供新的见解。Leut动态模型的质量将通过额外的EPR数据收集进行验证，这些数据将支持或驳斥预测的特定方面。拥有一个高质量的模型将允许Leut系统的结论为NSS家族提供重要的假设，这将是理解调节运输所必需的特定构象运动的中心。公共卫生相关性：对NSS家族如何管理交通有更好的了解将有助于开发更具体的治疗方法。其结果将是对大量精神和身体疾病有效且容易获得的药理化合物。

DESCRIPTION (provided by applicant): The serotonin, dopamine, and norepinephrine neurotransmitter transporters (SERT, DAT, and NET, respectively) are involved in a vast array of physical and mental health-related processes ranging from hypertension and heart failure to Parkinson's disease. SERT and DAT are also binding targets of cocaine and amphetamines. Elucidation of the major conformational changes these transporters undergo to regulate the flow of substrate would facilitate the development of pharmacological compounds to combat these ailments by providing an improved understanding of how these complex molecular machines function. SERT, DAT, and NET belong to the neurotransmitter sodium symporter (NSS) family of transporters. Currently, no high resolution structure exists for any member of the NSS family. However, a high resolution crystal structure for the bacterial homolog LeuT has provided insights into the structure and substrate binding of this important transporter family. However, while this crystal structure highlights a single closed- closed state in the transport cycle, multiple other conformations important for function remain unknown. The current work will utilize the NSS homolog LeuT crystal structure and electron paramagnetic resonance (EPR) distance and accessibility data in order to investigate how substrate passage is controlled through conformational changes. To achieve this end, a computational algorithm will be developed which will provide a continuous picture of LeuT as it progresses from one state to another. The second state will be described by the EPR data collected around the gating residues of LeuT. The algorithm will consist of an energy minimization process which is driven by the EPR data to reach the second conformation through a biologically relevant pathway. Such a method will provide a description of how LeuT changes structurally in order to control transport. The structural dynamics determined for LeuT will provide new insights into the homologous transporters of the NSS family. The quality of the dynamic model for LeuT will be validated through additional EPR data collection which will support or refute specific aspects of the prediction. Having a high quality model will allow conclusions for the LeuT system to provide important hypotheses for the NSS family which will be central to understanding the specific conformational motions that are necessary to regulate transport. PUBLIC HEALTH RELEVANCE: Having an improved understanding of how the NSS family regulates transport will facilitate the development of more specific therapeutics. The result will be pharmacological compounds which are potently effective and readily available for a large number of mental and physical diseases.