LeuT Membrane Transporter Dynamics Determined by EPR and Computational Techniques

由 EPR 和计算技术确定的 LeuT 膜转运蛋白动力学

• 批准号: 7902298
• 负责人: Nathan Alexander
• 金额: $ 2.99万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7902298
• 关键词: Algorithms; Amphetamines; Benchmarking; Binding; Binding Sites; Cocaine; Collaborations; Complex; Computational Technique; Computational algorithm; Crystallins; Data; Data Collection; Development; Disease; Dopamine; Education; Electron Spin Resonance Spectroscopy; Environment; Exposure to; Family; Heart failure; Homologous Gene; Hypertension; Ions; Leucine; Measurement; Measures; Mediating; Membrane; Membrane Proteins; Membrane Transport Proteins; Mental Health; Methods; Modeling; Molecular Conformation; Molecular Machines; Motion; Movement; Muramidase; Neurotransmitters; Norepinephrine; Parkinson Disease; Pathway interactions; Positioning Attribute; Process; Property; Protein Conformation; Proteins; Psyche structure; Relative (related person); Research; Research Infrastructure; Resolution; Resources; Serotonin; Side; Sodium; Solvents; Structure; System; Techniques; Therapeutic; Transport Process; Universities; Work; combat; comparative; flexibility; improved; insight; member; molecular dynamics; protein function; protein structure; protein structure prediction; public health relevance; reuptake; structural biology; symporter; three dimensional structure

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.

描述（由申请人提供）：5-羟色胺，多巴胺和去甲肾上腺素神经递质转运蛋白（分别是SERT，DAT和NET）参与了一系列与身体和心理健康有关的过程，范围从高血压和心脏病等性过程范围内到帕金森氏病。 SERT和DAT也是可卡因和苯丙胺的结合靶标。阐明主要构象变化这些转运蛋白会进行调节底物的流动，将促进药理学化合物的发展，通过对这些复杂的分子机器的功能有了深入的了解，以对抗这些疾病。 SERT，DAT和NET属于转运蛋白的神经递质钠共钠（NSS）家族。目前，NSS家族的任何成员都不存在高分辨率结构。但是，细菌同源物Leut的高分辨率晶体结构为该重要转运蛋白家族的结构和底物结合提供了见解。但是，尽管这种晶体结构突出显示了传输周期中单个闭合状态，但对功能的多个其他构构仍然未知。当前的工作将利用NSS同源物Leut晶体结构和电子顺磁共振（EPR）距离和可访问性数据，以研究如何通过构象变化来控制底物段。为了实现这一目的，将开发一种计算算法，该算法将在Leut从一个状态发展到另一个状态时提供连续的图片。第二个状态将由Leut门控残基收集的EPR数据描述。该算法将由一个能量最小化过程组成，该过程由EPR数据驱动，以通过生物学相关的途径达到第二构象。这种方法将提供有关Leut在结构上如何变化以控制运输的描述。确定Leut的结构动力将为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其他EPR数据收集来验证，该数据将支持或反驳预测的特定方面。拥有高质量的模型将使Leut系统的结论为NSS家族提供重要的假设，这对于了解调节运输所需的特定构象动作至关重要。公共卫生相关性：对NSS家族如何调节运输方式有了改进的了解，将有助于开发更具体的治疗疗法。结果将是药理学化合物，这些化合物有效，可用于大量的精神和身体疾病。