LeuT Membrane Transporter Dynamics Determined by EPR and Computational Techniques

描述（由申请者提供）：血清素、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、心力衰竭到帕金森病等一系列身体和精神健康相关的过程。SERT和DAT也是可卡因和安非他明的结合靶点。阐明这些转运体在调节底物流动中所经历的主要构象变化，将有助于通过更好地了解这些复杂分子机器的功能，促进药物化合物的发展，以对抗这些疾病。SERT、DAT和NET属于神经递质钠同向转运蛋白（NSS）家族。目前，NSS家族的任何成员都没有高分辨率的结构。然而，细菌同源物LeuT的高分辨率晶体结构为这一重要转运蛋白家族的结构和底物结合提供了见解。然而，虽然这种晶体结构在传递循环中强调了单一的封闭状态，但对功能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如何通过构象变化控制衬底通过。为了实现这一目标，将开发一种计算算法，该算法将提供LeuT从一种状态发展到另一种状态的连续图像。第二种状态将通过在LeuT的门控残基周围收集的EPR数据来描述。该算法将由能量最小化过程组成，该过程由EPR数据驱动，通过生物学相关途径达到第二种构象。这种方法将提供LeuT如何在结构上改变以控制传输的描述。确定LeuT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数据收集来验证，这些数据将支持或反驳预测的特定方面。拥有一个高质量的模型将使LeuT系统的结论能够为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核心。公共卫生相关性：提高对NSS家族如何调节转运的理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易于获得的药理学化合物。

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.