Investigating the role of Substrate Binding in LeuT Transport With RosettaEPR

描述（由申请人提供）：尽管它们在自然界中普遍存在，并作为药物和治疗靶点，但许多膜蛋白继续逃避x射线晶体学和核磁共振的结构测定。定位自旋标记与电子顺磁共振（SDSL-EPR）的结合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进行研究。然而，SDSL-EPR不能直接产生高分辨率结构。目前的提案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限制将与基于蒙特卡罗的计算方法相结合，用于蛋白质的从头结构预测。在从EPR实验数据中开发出基于知识的电位后，将使用模拟和真实EPR数据对已知结构的蛋白质进行基准测试。此外，ROSETTAEPR和EPR实验距离和可及性数据将用于确定参与亮氨酸运输的LeuT载脂蛋白、Na+和Na+/亮氨酸结合的结构中间体。LeuT是神经递质钠同向转运蛋白（NSS）家族的细菌同源物，其中包括多巴胺，血清素和去甲肾上腺素转运蛋白。虽然没有NSS转运体的高分辨率结构，但LeuT的细胞外底物结合构象已经通过x射线晶体学确定。然而，目前的结构是LeuT传输周期的静态快照；此外，它们被认为是以一种潜在的抑制形式捕获的。因此，EPR光谱已被用来阐明蛋白质的动力学。研究发现，Na+结合导致细胞外环中蛋白质柔韧性的增加和底物渗透途径的水化，而随后亮氨酸的结合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. 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.