Structure-Function relation of Connexin disease mutations

描述(由申请人提供)：N-末端结构域(NT，残基1-22)是连接蛋白通道的膜选择性和电压依赖门控的重要决定因素，也是两种常见遗传病的敏感突变靶点：X连锁Charcot-Marie-Tooth(Cx32)和非综合征和综合征耳聋(Cx26)。这项提议将通过应用协同计算和实验方法来确定Cx32和Cx26的NT致病突变如何改变通道功能和通道生物合成。野生型和引起NT突变的疾病之间的功能差异被认为是由于通道结构的特定变化引起的。这项研究将检查9个包含Cx32和Cx26突变的NT基因座。在某些情况下，同一基因座的突变会改变Cx26和Cx32通道的不同功能，这表明相同或同源的氨基酸替换会导致这两个连接蛋白的不同结构缺陷。研究将以Cx26半通道和Cx32同源模型的晶体结构为指导，这两个模型都由全原子分子动力学(MD)模拟改进，并被证明与生物开放通道的结构密切对应。本研究将利用2D核磁共振技术解决突变的NT多肽的结构问题。膜环境中较长的野生型和突变型多肽(NT-CL结构域，残基1-114)的三维核磁共振成像和X射线结晶学组装通道的结构溶液已经被启动。由此得到的连接蛋白通道的原子模型将通过全原子MD模拟进行改进，通过计算确定对离子和第二信使的通透性，并与实验进行比较。这一实验策略提供了对原子模型准确性的敏感测试，对渗透选择性的分子机制的洞察以及这些机制是如何因突变而改变的，以及结构-功能关系的可测试假说。这项研究将通过确定连接蛋白亚基插入到犬微粒体膜中的位置和稳定性，NT在亚单位齐聚中的作用，以及NT在质膜插入之前何时以及如何在组装的半管孔中的最终位置来研究NT在通道生物发生中的作用。并行计算研究将提供一个严格的机制框架来指导这些实验研究。这一新的基础知识将为理解导致NT突变的一类疾病的分子缺陷提供一个框架，这种突变不是质膜插入的，而是被困在细胞质隔室中，并以降解为目标。该项目具有高度的协作性，将调查人员与 在连接蛋白通道的结构确定、计算方法和生物物理表征方面具有成熟的专业知识。这些结果将为阐明缝隙连接蛋白病的病因学和开发有效的治疗方法提供新的信息基础。

DESCRIPTION (provided by applicant): The N-terminal domain (NT, residues 1-22) is an important determinant of perm-selectivity and voltage-dependent gating of connexin channels and a sensitive mutational target underlying two common inherited diseases: X-linked Charcot-Marie-Tooth (Cx32) and nonsyndromic and syndromic deafness (Cx26). This proposal will determine how disease causing mutations in the NT of Cx32 and Cx26 alter channel function and channel biosynthesis by applying synergistic computational and experimental approaches. Differences in function between wild type and disease causing NT mutations are hypothesized to arise from specific changes in channel structure. The study will examine 9 NT loci comprising mutations in both Cx32 and Cx26. In several cases, mutations of the same locus alter Cx26 and Cx32 channel function differently, suggesting that identical or homologous amino acid substitutions cause different structural defects in the two connexins. Studies will be guided by the crystal structure of a Cx26 hemichannel and a Cx32 homology model, both refined by all-atom molecular dynamics (MD) simulation and shown to closely correspond to the structure of the biological open channel. The study will solve the structure of mutant NT peptides by 2D NMR. Structural solutions of longer wild-type and mutant peptides (NT-CL domain, residues 1-114) in a membrane environment by 3D NMR, and assembled channels by x-ray crystallography have been initiated. Resulting atomic models of connexin channels will be refined by all-atom MD simulations, the permeabilities to ions and second messengers determined computationally and compared to experimental. This experimental strategy provides a sensitive test of the accuracy of atomic models, insights into molecular mechanisms of perm-selectivity and how these are changed by mutation, as well as testable hypotheses of structure-function relations. The study will investigate the role of the NT in channel biogenesis by determining the position and stability of the NT of connexin subunits inserted into canine microsomal membranes, the role of the NT in subunit oligomerization, and when and how the NT assumes its final position deep within the pore of assembled hemichannels prior to plasma membrane insertion. Parallel computational studies will provide a rigorous mechanistic framework that will guide these experimental studies. This new, fundamental knowledge will provide a framework for understanding the molecular defects of the class of disease causing NT mutations that are not plasma membrane inserted but trapped in cytosolic compartments and targeted for degradation. The project is highly collaborative, bringing together investigators with proven expertise in structural determination, computational methods and biophysical characterization of connexin channels. The results will provide new information fundamental to the elucidation of connexin disease etiology and to the development of effective treatments.