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：钠同向转运体（NSS）转运体家族的功能动力学的理解。NSS成员包括人类多巴胺、5-羟色胺和去甲肾上腺素转运蛋白，它们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中。这种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启动细胞进行后续信号传导事件至关重要。NSS参与情绪、奖赏和运动的调节，并且与中枢神经系统疾病如抑郁症、焦虑症、自闭症、癫痫、注意缺陷多动障碍和强迫症以及对非法药物如可卡因和安非他明的滥用和成瘾有关。在这项建议的调查将集中在亮氨酸转运蛋白（LeuT），细菌的NSS家族成员和卓越的模型，了解NSS功能。虽然先前的研究已经开始阐明LeuT的结构和动力学，但与构象循环相关的关键问题仍然没有答案。这个建议的具体目标将解决这些问题中最关键的：需要什么样的螺旋运动来产生细胞内渗透途径？这条路在哪里？LeuT的机制相关的开放构象是什么？解决这些问题将是根本的了解运输机制的LeuT，这仍然是有争议的。这些目标的设计将测试由海因转运蛋白（Mhp1）结构家族支持的假设，即LeuT通过摇杆开关机制发挥作用。这种机制意味着一个刚性的身体重新定位的伪二聚体基序的LeuT，允许访问的主要底物结合位点从膜的两侧。为了解决这些问题，电子顺磁共振（EPR）光谱和计算建模将被用来定义的性质和幅度涉及LeuT运输的构象变化。定点自旋标记（SDSL）和EPR提供了强有力的工具来描述全球动态运动，以及在残基环境的变化，局部构象变化的指示。此外，EPR测量将使用从头蛋白质折叠算法Rosetta生成开放构象的第一个经验模型。这里提出的工作将提供新的见解LeuT和NSS运输的构象动力学。此外，在这项工作中提出的EPR光谱和计算结构测定的混合技术的应用将代表在膜蛋白结构测定领域的一个显着的方法学进步。

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.