Conformational Changes Underlying Intracellular Gating of the Leucine Transporter

描述（由申请人提供）：本提案的长期目标是促进对神经递质：钠同向转运（NSS）转运蛋白家族的功能动力学的理解。NSS成员包括人类多巴胺、血清素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再摄取到突触前神经元和神经胶质。该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随后的信号传导事件启动细胞至关重要。nss参与情绪、奖励和运动的调节，并与中枢神经系统疾病有关，如抑郁、焦虑、自闭症、癫痫、注意缺陷多动障碍、强迫症以及滥用和成瘾非法药物，如可卡因和安非他明。本提案的研究将集中在亮氨酸转运蛋白（LeuT）上，它是NSS家族的细菌成员，也是理解NSS功能的卓越模型。虽然以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。本提案的具体目标将解决这些问题中最关键的问题：需要什么样的螺旋运动来产生细胞内渗透途径？这条通道位于哪里？LeuT的开放构象是什么？回答这些问题将是理解仍有争议的LeuT输运机制的基础。这些目标的设计将验证由hydrantoin transporter （Mhp1）家族结构支持的假设，即LeuT通过摇杆开关机制起作用。这一机制意味着LeuT伪二聚体基元的刚体重定向，允许从膜的两侧进入初级底物结合位点。为了解决这些问题，电子顺磁共振（EPR）光谱和计算模型将被用来定义轻中子输运中构象变化的性质和幅度。位点定向自旋标记（SDSL）和EPR为描述整体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测量将使用从头开始的蛋白质折叠算法Rosetta来生成开放构象的第一个经验模型。本文提出的工作将为LeuT和NSS输运的构象动力学提供新的见解。此外，本文提出的EPR光谱与计算结构测定混合技术的应用将是膜蛋白结构测定领域的重要方法进步。

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.