Cavities in Choline Acetyltransferase and Neuromuscular Disorders

描述（由申请人提供）：我们的目标是了解胆碱乙酰转移酶（ChAT）先天性突变的影响，该酶合成原型神经递质乙酰胆碱。ChAT活性的降低与许多神经系统疾病状态相关，并且该酶的突变导致称为先天性肌无力综合征伴阵发性呼吸暂停（CMS-EA）的严重神经肌肉疾病。我们的晶体结构的ChAT表明，已知的先天性突变广泛分布在酶，尽管它们的共同作用的功能，和最近的结构表明，两个突变影响构象的催化组氨酸残基。此外，ChAT中的核残基填充差，留下异常大量的空腔（空隙）。基于这些观察结果，我们假设ChAT中大量的空腔使其构象不稳定，因此点突变导致结构变化，从而破坏功能。我们将使用结构，生物物理和细胞生物学方法的组合来测试这一假设，重点是在两个先天性突变附近的一个空腔，其结构特征。提出了两个具体目标：1）表征内腔在介导两种先天性突变的功能效应中的作用，2）评估填充腔如何影响与先天性突变相关的结构变化。这项工作将作为我们关于ChAT中包装缺陷作用的假设的初步测试， 直接解决人类疾病的分子机制，并指导未来的研究ChAT和运动障碍与其功能下降。

DESCRIPTION (provided by applicant): Our goal is to understand the effects of congenital mutations in choline acetyltransferase (ChAT), which synthesizes the prototypical neurotransmitter acetylcholine. Decreases in ChAT activity are associated with a number of neurological disease states, and mutations in the enzyme cause a severe neuromuscular disorder known as congenital myasthenic syndrome with episodic apnea (CMS-EA). Our crystal structure of ChAT showed that the known congenital mutations are distributed widely over the enzyme despite their common effects on function, and recent structures show that two of the mutations affect the conformation of the catalytic histidine residue. Further, the core residue packing in ChAT is poor, leaving an unusually large number of cavities (voids). Based on these observations, we hypothesize that the large number of cavities in ChAT makes it conformationally unstable, so that point mutations cause structural changes that propagate to disrupt function. We will use a combination of structural, biophysical, and cell biological approaches to test this hypothesis focusing on a cavity near the two congenital mutations that have been characterized structurally. Two specific aims are proposed: 1) characterize the role of an internal cavity in mediating the functional effects of two congenital mutations, 2) assess how filling the cavity effects structural changes associated with the congenital mutations This work will serve as an initial test of our hypothesis regarding the role of packing defects in ChAT, directly addressing the molecular mechanism underlying a human disease and guiding future studies of ChAT and the motor disorders associated with decreases in its function.