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Structure-Function relation of Connexin disease mutations

连接蛋白疾病突变的结构-功能关系

基本信息

批准号：
9189954
负责人：
Thaddeus Andrew Bargiello
金额：
$ 29.94万
依托单位：
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-01 至 2017-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9189954
关键词：
Structure Function relation Connexin disease

项目摘要

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.

描述（由申请人提供）：N 端结构域（NT，残基 1-22）是连接蛋白通道通透选择性和电压依赖性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 连锁夏科-马里-图思 (Cx32) 和非综合征性和综合征性耳聋 (Cx26)。该提案将通过应用协同计算和实验方法来确定 Cx32 和 Cx26 NT 中引起疾病的突变如何改变通道功能和通道生物合成。野生型和引起疾病的 NT 突变之间的功能差异被假设是由通道结构的特定变化引起的。该研究将检查包含 Cx32 和 Cx26 突变的 9 个 NT 位点。在一些情况下，同一位点的突变会以不同的方式改变 Cx26 和 Cx32 通道功能，这表明相同或同源的氨基酸取代会导致两个连接蛋白出现不同的结构缺陷。研究将以 Cx26 半通道和 Cx32 同源模型的晶体结构为指导，这两个模型均通过全原子分子动力学 (MD) 模拟进行细化，并显示与生物开放通道的结构密切对应。该研究将通过 2D NMR 解析突变型 NT 肽的结构。已经开始通过 3D NMR 在膜环境中对较长的野生型和突变肽（NT-CL 结构域，残基 1-114）进行结构解，并通过 X 射线晶体学进行组装通道。由此产生的连接蛋白通道的原子模型将通过全原子MD模拟、通过计算确定并与实验进行比较确定的离子和第二信使的渗透性来完善。该实验策略提供了对原子模型准确性的敏感测试，深入了解选择性的分子机制以及突变如何改变这些机制，以及结构-功能关系的可测试假设。该研究将通过确定插入犬微粒体膜的连接蛋白亚基的NT的位置和稳定性、NT在亚基寡聚化中的作用，以及NT在质膜插入之前何时以及如何在组装的半通道的孔深处占据最终位置，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引起 NT 突变的疾病类别的分子缺陷提供一个框架，这些缺陷不是插入质膜而是被困在细胞质区室中并被靶向降解。该项目是高度协作的，将研究人员聚集在一起在连接蛋白通道的结构测定、计算方法和生物物理表征方面拥有经过验证的专业知识。研究结果将为阐明连接蛋白疾病的病因学和开发有效的治疗方法提供基础的新信息。