Structure-Function relation of Connexin disease mutations

连接蛋白疾病突变的结构-功能关系

• 批准号: 8725194
• 负责人: Thaddeus Andrew Bargiello
• 金额: $ 30.13万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8725194
• 关键词: Address; Amino Acid Substitution; Amino Acids; Anabolism; Antibodies; Biogenesis; Biological; Canis familiaris; Cell Line; Cell membrane; Cellular biology; Charcot-Marie-Tooth Disease; Collaborations; Computer Simulation; Computing Methodologies; Connexins; Crystallography; Cysteine; Defect; Development; Disease; Environment; Etiology; Failure; Fluorescence Spectrometry; Genes; Homology Modeling; Human; Immunoprecipitation; Inherited; Ions; Knowledge; Link; Maps; Measures; Membrane; Methods; Missense Mutation; Modeling; Modification; Molecular; Mono-S; Monoclonal Antibodies; Mutation; N-terminal; Pathway interactions; Peptides; Permeability; Phenotype; Plasma; Positioning Attribute; Probability; Process; Property; Research Personnel; Role; Second Messenger Systems; Solutions; Structural Models; Structure; Sulfhydryl Compounds; Testing; Validation; base; biophysical properties; computer studies; deafness; disease-causing mutation; effective therapy; experience; flexibility; gain of function; insight; loss of function; loss of function mutation; molecular dynamics; mutant; prevent; research study; second messenger; skin disorder; therapeutic development; trafficking; voltage

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）是连接蛋白通道的热选择性和电压依赖性门控的重要决定因素，也是两种常见遗传性疾病的敏感突变靶点：x连锁沙克-玛丽- tooth （Cx32）和非综合征性和综合征性耳聋（Cx26）。该提案将通过协同计算和实验方法确定Cx32和Cx26 NT的致病突变如何改变通道功能和通道生物合成。野生型和疾病引起的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。该研究将检测包含Cx32和Cx26突变的9个NT位点。在一些情况下，相同位点的突变改变了Cx26和Cx32通道的不同功能，这表明相同或同源的氨基酸取代导致了两种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，这两个模型都是通过全原子分子动力学（MD）模拟改进的，并显示出与生物开放通道的结构密切对应。本研究将利用二维核磁共振技术解析突变体NT肽的结构。通过三维核磁共振在膜环境中对较长的野生型和突变型肽（NT-CL结构域，残基1-114）进行了结构解决，并通过x射线晶体学对组装通道进行了研究。由此产生的连接蛋白通道的原子模型将通过全原子MD模拟来完善，对离子和第二信使的渗透率将通过计算确定并与实验进行比较。该实验策略为原子模型的准确性提供了一个敏感的测试，深入了解了分子选择性的机制，以及这些机制如何被突变改变，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确定其最终位置，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，这些疾病不是插入质膜而是被困在细胞质室中并被降解。该项目是高度协作的，汇集了研究人员与