Investigating the role of Substrate Binding in LeuT Transport With RosettaEPR

描述(申请人提供)：尽管它们在自然界中普遍存在，并且作为药物和治疗靶点，许多膜蛋白继续躲避X射线结晶学和核磁共振的结构测定。结合定点自旋标记和电子顺磁共振(SDSL-EPR)的方法正成为膜蛋白结构表征的一种日益流行的方法，因为它们可以相对容易地被研究。然而，SDSL-EPR不能直接产生高分辨率结构。目前的提议描述了一种新的方法，ROSETTAEPR，以克服这一障碍。ROSETTAEPR将是一个工具包，其中由EPR确定的距离和可及性约束将与基于蒙特卡洛的计算方法相结合，用于蛋白质从头结构预测。在从EPR实验数据中开发出基于知识的势之后，它将使用模拟和真实的EPR数据以已知结构的蛋白质为基准。此外，ROSETTAEPR和EPR实验距离和可及性数据将用于确定参与亮氨酸运输的Leut apo、Na+和Na+/Leucine结合的结构中间体。Leut是神经递质钠转运体(NSS)蛋白家族的细菌同系物，包括多巴胺、5-羟色胺和去甲肾上腺素转运体。虽然目前还没有NSS转运蛋白的高分辨结构，但Leut的面向细胞外底物结合的构象已经通过X射线结晶学确定。然而，目前的结构是Leut运输周期的静态快照；此外，它们被认为是以一种可能被抑制的形式捕获的。因此，电子顺磁共振波谱被用来阐明蛋白质的动力学。结果发现，Na+结合导致细胞外环蛋白质弹性增加和底物渗透途径水化，而随后的亮氨酸结合导致细胞外前庭关闭并变得僵硬。ROSETTAEPR将允许在低分辨率EPR数据的基础上对这些中间体进行高分辨率结构解析。

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.