Investigating the role of Substrate Binding in LeuT Transport With RosettaEPR

使用 RosettaEPR 研究底物结合在 LeuT 转运中的作用

• 批准号: 8257632
• 负责人: Stephanie Judith Han Hirst DeLuca
• 金额: $ 2.67万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8257632
• 关键词: Anxiety; Benchmarking; Binding; Binding Sites; Biochemical; Biological Models; Brain; Cells; Central Nervous System Diseases; Characteristics; Complex; Computing Methodologies; Data; Development; Disadvantaged; Dopamine; Drug Delivery Systems; Electron Spin Resonance Spectroscopy; Epilepsy; Family; Functional disorder; Homologous Gene; Hybrids; Hydration status; Internet; Ions; Lead; Leucine; Light; Link; Locomotion; Measures; Membrane; Membrane Proteins; Mental Depression; Mental disorders; Methods; Molecular; Molecular Conformation; Moods; Movement; Muramidase; Nature; Neurotransmitters; Nuclear Magnetic Resonance; Obsessive-Compulsive Disorder; Pathway interactions; Play; Prevalence; Process; Protein Dynamics; Protein Family; Proteins; Relative (related person); Resolution; Rewards; Rhodopsin; Role; Serotonin; Simulate; Site; Sodium; Spin Labels; Structural Models; Structure; Testing; Therapeutic Agents; Travel; Vestibule; X-Ray Crystallography; arm; base; dopamine transporter; extracellular; flexibility; knowledge base; member; neurotransmitter reuptake; noradrenaline transporter; programs; protein folding; protein function; protein structure; protein structure prediction; relating to nervous system; restraint; serotonin transporter; simulation; symporter; therapeutic target; tool

DESCRIPTION (provided by applicant): Despite their prevalence in nature and as drug and therapeutic targets, many membrane proteins continue to evade structure determination by X-ray crystallography and NMR. The combination of site-directed spin labeling with electron paramagnetic resonance (SDSL-EPR) is becoming an increasingly popular method for the structural characterization of membrane proteins due to the relative ease with which they can be studied. However, SDSL-EPR does not yield high-resolution structures directly. The current proposal describes a new method, ROSETTAEPR, to overcome this obstacle. ROSETTAEPR will be a toolkit in which distance and accessibility restraints determined by EPR will be combined with Monte Carlo-based computational methods for the de novo structure prediction of proteins. After developing knowledge-based potentials derived from EPR experimental data, it will be benchmarked on proteins of known structure using both simulated and real EPR data. In addition, ROSETTAEPR and EPR experimental distance and accessibility data will be used to determine the LeuT apo, Na+, and Na+/leucine bound structural intermediates involved in leucine transport. LeuT is a bacterial homolog of the neurotransmitter sodium symporter (NSS) protein family, which includes the dopamine, serotonin, and norepinephrine transporters. While there are no high-resolution structures of the NSS transporters, extracellular-facing substrate-bound conformations of LeuT have been determined by X-ray crystallography. However, the current structures are static snapshots of the LeuT transport cycle; furthermore, they are believed to have been captured in a potentially inhibited form. Therefore, EPR spectroscopy has been employed to shed light on the dynamics of the protein. It was found that Na+ binding causes an increase in protein flexibility in the extracellular loops and hydration of the substrate permeation pathway, while subsequent binding of leucine causes the extracellular vestibule to close and become rigid. ROSETTAEPR will allow for the high-resolution structural elucidation of these intermediates based on low-resolution EPR data. PUBLIC HEALTH RELEVANCE: The dysfunction of neurotransmitter sodium symporter (NSS) proteins is a common characteristic of central nervous system (CNS) diseases, such as depression, anxiety, obsessive compulsive disorder (OCD), and epilepsy. Understanding how these proteins function on a structural level will aid in the development of new, more effective therapeutic agents that specifically target neural processes underlying mood, reward, and locomotion.

描述（由申请人提供）：尽管它们在自然界中流行，并且作为药物和治疗靶标，但许多膜蛋白仍继续通过X射线晶体学和NMR逃避结构。由于可以研究它们的相对容易性，因此将位置定向的自旋标记与电子顺磁共振（SDSL-EPR）的结合成为膜蛋白结构表征的越来越流行的方法。但是，SDSL-EPR不会直接产生高分辨率结构。当前的提案描述了一种新方法Rosettaepr来克服这一障碍。 Rosettaepr将是一种工具包，在该工具包中，EPR确定的距离和可及性约束将与基于Monte Carlo的基于蛋白质的从头结构预测的计算方法结合使用。在开发了来自EPR实验数据的基于知识的电位之后，它将使用模拟和实际EPR数据基于已知结构的蛋白质进行基准测试。此外，RosettaEPR和EPR实验距离和可访问性数据将用于确定列氨酸转运涉及的Leut Apo，Na+和Na+/Leucine结合的结构中间体。 Leut是神经递质钠共糖（NSS）蛋白家族的细菌同源物，其中包括多巴胺，5-羟色胺和去甲肾上腺素转运蛋白。尽管没有NSS转运蛋白的高分辨率结构，但已通过X射线晶体学确定了面向细胞外底物结合的Leut的构象。但是，当前的结构是Leut运输周期的静态快照。此外，据信它们是以潜在抑制形式捕获的。因此，EPR光谱已被用于阐明蛋白质的动力学。发现Na+结合会导致细胞外环的蛋白质柔韧性和底物渗透途径的水合，而亮氨酸的随后结合会导致细胞外前庭关闭并变得刚性。 RosettaEPR将允许基于低分辨率EPR数据对这些中间体进行高分辨率结构性阐明。 公共卫生相关性：神经递质钠钠（NSS）蛋白的功能障碍是中枢神经系统（CNS）疾病的常见特征，例如抑郁，焦虑，强迫症（OCD）和癫痫病。了解这些蛋白质在结构水平上的作用将如何有助于开发新的，更有效的治疗剂，这些治疗剂专门针对情绪，奖励和运动的神经过程。