Conformational Changes Underlying Intracellular Gating of the Leucine Transporter

描述(由申请人提供)：这项建议的长期目标是促进对神经递质：钠转运体(NSS)转运体家族功能动力学的理解。NSS成员包括人多巴胺、5-羟色胺和去甲肾上腺素转运体，利用钠和氯梯度促进神经递质从突触重新摄取到突触前神经元和神经胶质细胞。这一功能对于终止神经化学信号、维持细胞内神经递质浓度以及为后续信号事件准备细胞至关重要。NSSS参与情绪、奖励和运动的调节，并与抑郁症、焦虑症、自闭症、癫痫、注意力缺陷多动障碍和强迫症等中枢神经系统疾病以及对可卡因和苯丙胺等非法药物的滥用和上瘾有关。这项提案中的研究将集中在亮氨酸转运体(Leut)，它是NSS家族的细菌成员，也是了解NSS功能的卓越模型。虽然先前的研究已经开始阐明Leut的结构和动力学，但与构象周期相关的关键问题仍未得到解答。这项提案的具体目标将解决这些问题中最关键的问题：需要什么螺旋运动才能产生细胞内渗透途径？这条小路在哪里？在力学上相关的Leut开放构象是什么？回答这些问题将是理解Leut的运输机制的基础，Leut的运输机制仍然存在争议。这些AIMS的设计将检验这一假说，该假说得到了海因转运蛋白(Mhp1)家族结构的支持，即Leut通过摇杆开关机制发挥作用。这一机制意味着Leut的伪二聚体基序的刚体重定向，允许从膜的两侧进入初级底物结合部位。为了解决这些问题，电子顺磁共振(EPR)波谱和计算模型将被用来定义Leut运输中涉及的构象变化的性质和幅度。定点定向自旋标记(SDSL)和EPR为描述全球动态运动以及指示局部构象变化的残基环境变化提供了强大的工具。此外，EPR测量将被用于使用从头蛋白质折叠算法Rosetta生成第一个开放构象的经验模型。本文提出的工作将对Leut和NSS运输的构象动力学提供新的见解。此外，本文提出的EPR谱与计算结构测定的混合技术的应用将是膜蛋白质结构测定领域的一项重大的方法学进步。 公共卫生相关性：该提案的目标是了解神经递质转运体的功能。这些蛋白质与许多神经疾病有关，包括抑郁症、自闭症和癫痫，以及对非法药物的滥用和上瘾。这项工作将为治疗这些慢性病的药物的开发提供信息。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to advance the understanding of the functional dynamics of the neurotransmitter:sodium symporter (NSS) family of transporters. NSS members including human dopamine, serotonin, and norepinephrine transporters harness sodium and chloride gradients to facilitate reuptake of neurotransmitters from the synapse into presynaptic neurons and glia. This function is vital for terminating neurochemical signals, maintaining intracellular neurotransmitter concentrations, and priming the cell for subsequent signaling events. NSSs participate in the regulation of mood, reward, and locomotion and have been implicated in diseases of the central nervous system such as depression, anxiety, autism, epilepsy, attention deficit hyperactivity disorder, and obsessive compulsive disorder as well as abuse and addiction to illicit drugs such as cocaine and amphetamine. Investigations in this proposal will focus on the leucine transporter (LeuT), a bacterial member of the NSS family and the preeminent model for understanding NSS function. While previous studies have begun to elucidate the structure and dynamics of LeuT, key questions related to the conformational cycle remain unanswered. The specific aims of this proposal will address the most critical of these questions: What helical motions are required to generate an intracellular permeation pathway? Where is this pathway located? What is the mechanistically relevant open-in conformation of LeuT? Answering these questions will be fundamental to understanding the transport mechanism of LeuT, which remains controversial. The design of these aims will test the hypothesis, supported by the hydrantoin transporter (Mhp1) family of structures, that LeuT functions by a rocker switch mechanism. This mechanism implies a rigid body reorientation of the pseudo-dimer motifs of LeuT that allows access to the primary substrate binding site from either side of the membrane. To address these questions, electron paramagnetic resonance (EPR) spectroscopy and computational modeling will be employed to define the nature and amplitude of conformational changes involved in LeuT transport. Site-directed spin labeling (SDSL) and EPR provide powerful tools to describe global dynamic motion as well as changes in residue environments indicative of local conformational changes. Furthermore, EPR measurements will be employed to generate the first empirical model of the open-in conformation using the de novo protein folding algorithm Rosetta. The work proposed here will provide novel insights into the conformational dynamics of LeuT and NSS transport. Furthermore, application of the hybrid technique of EPR spectroscopy and computational structure determination proposed in this work will represent a significant methodological advance in the field of membrane protein structure determination. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to understand the function of neurotransmitter transporters. These proteins are involved in a host of neurological disorders, including depression, autism, and epilepsy, as well as abuse and addiction to illicit drugs. This work will inform the development of drugs to treat these chronic conditions.