The Role of Zinc ions for RNA Binding and Catalytic Function of the DYW-deaminase

锌离子对 RNA 结合的作用和 DYW 脱氨酶的催化功能

• 批准号: 9280248
• 负责人: Michael Lloyd Hayes
• 金额: $ 14.6万
• 依托单位: CALIFORNIA STATE UNIVERSITY LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-12 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9280248
• 关键词: Amino Acid Substitution; Amino Acids; Antibody Diversity; Binding; Binding Sites; Biological Assay; C-terminal; Catalysis; Cell physiology; Chloroplasts; Crystallization; Crystallography; Cytidine; Cytidine Deaminase; DNA; Data; Deaminase; Deamination; Edetic Acid; Endoribonucleases; Enzymes; Event; Future; Glutamates; Goals; Hydrolase; Hydroxyl Radical; In Vitro; Ions; Ligand Binding; Ligands; Link; Location; Measures; Metal Binding Site; Metals; Mitochondria; Modeling; Mutation; Nucleic Acid Binding; Nucleic Acids; Nucleotide Deaminases; Nucleotides; Plastids; Proteins; Protons; Pyrimidine; RNA; RNA Binding; RNA Editing; Reaction; Recombinant Proteins; Recombinants; Ribonucleases; Ribonucleotides; Role; Series; Site; Specificity; Structure; Surveys; System; Testing; Transcript; Vascular Plant; Viral; Zinc; analog; experimental study; in vitro activity; innovation; metal chelator; new therapeutic target; novel; stoichiometry

Project Summary/Abstract RNA editing and cleavage of chloroplast transcripts require proteins with the DYW-deaminase domain. The DYW-deaminase shares several features common to nucleotide deaminases and comprises the enzymatic portion of the RNA editing machinery. Some DYW-deaminase proteins are not associated with any RNA editing function and are required for ribonuclease cleavage. On this basis the domain can be broken down into one class with RNA editing functions and a second class with ribonuclease functions. Related enzymes function exclusively as amino hydrolases and are critical for various cellular functions including RNA editing, antibody diversity, and viral defense. Each DYW-deaminase domains binds two zinc ions. Zinc #1 is most likely coordinated by the typical C/HAE---CXXC motif found in all nucleotide deaminases. Amino acids in a unique C- terminus likely coordinate Zinc #2 and are nearby the catalytic zinc ion binding site. A general mechanism for cytidine deamination does not require two zinc ions. The second zinc ion could have a unique co-catalytic function, serve as a structural feature, or have a role in ligand binding. Zinc ions are not required for all ribonuclease mechanisms and addition of EDTA promotes the ribonuclease cleavage activity of recombinant DYW-deaminases in vitro. One or both zinc ions might operate as a zinc “switch” that governs the deaminase and ribonuclease functions of the domain. This project aims to investigate a model where zinc ions act as a “switch” that determines catalytic function. Zinc binding in DYW-deaminases with endoribonuclease and RNA editing functions will be compared to investigate if amino acid differences present in each functional classes determine zinc stoichiometry. A pool of recombinant DYW-deaminases with amino acid substitutions in putative zinc coordinating residues will be created to pinpoint the location of zinc coordinating residues. Endoribonuclease activities will be assayed for rDYW-deaminase proteins with known RNA editing functions as well as known endoribonuclease functions in the presence or absence of metal chelators. Ribonuclease activity will be measured for recombinant proteins with altered zinc stoichiometries and mutations in zinc coordinating residues. A series of proteins with amino acid changes might discover a link between zinc binding and enzymatic function as a well as to establish the reaction mechanism for RNA cleavage. An activity assay for RNA editing that includes addition of exogenous DYW-deaminases will be developed. The activity of DYW-deaminases with different zinc stiochiometries will be measured to determine the relationship between zinc ion binding and aminohydrolase activity. The second zinc ion may have a specialized role in ligand binding and to test this possibility the nucleic acid ligand requirements of the DYW-deaminase will be determined using RNAs and putative substrates with modified nucleotides. Finally, crystallization conditions for recombinant proteins will be screened. Crystals could be used in future experiments aimed at solving the structure of the DYW-deaminase.

项目摘要/摘要 叶绿体转录本的RNA编辑和切割需要具有DYW - 脱氨酶结构域的蛋白质。这 DYW - 脱氨酶具有核苷酸脱氨酶共有的几种特征，并包括酶促 RNA编辑机械的一部分。某些DYW - 脱氨基酶蛋白与任何RNA无关 编辑功能，是核糖核酸酶清洁所必需的。在此基础上，域可以分解为 一个具有RNA编辑功能的类和带有核糖核酸酶功能的第二类。相关酶 功能仅作为氨基水解酶，对于包括RNA编辑在内的各种细胞功能至关重要， 抗体多样性和病毒防御。每个DYW脱氨酶结构域结合了两个锌离子。锌＃1很可能是 由在所有核苷酸脱氨酶中发现的典型c/hae协调的--- CXXC基序。独特的C-中的氨基酸 末端可能会协调锌＃2，并且在催化锌离子结合位点附近。一种通用机制 胞苷死亡不需要两个锌离子。第二个锌离子可以具有独特的共催化 功能，充当结构特征，或在配体结合中起作用。所有人都不需要锌离子 核糖核酸酶机制和EDTA的添加促进了重组的核糖核酸酶清洁活动 体外DYW脱氨酶。一个或两个锌离子可能作为控制脱氨酶的锌“开关” 和域的核糖核酸酶功能。 该项目旨在调查锌离子作为决定催化的“开关”的模型 功能。将比较具有内核核酸酶和RNA编辑功能的DYW脱氨酶结合的锌结合 为了研究每个功能类别中是否存在氨基酸差异确定锌化学计量。游泳池 在假定的锌配位保留中，重组dyw脱氨酶与氨基酸取代将是 创建以查明锌协调残差的位置。内切核酸酶活动将被分配 具有已知RNA编辑函数的RDYW - 脱氨基酶蛋白以及已知的内核酸酶函数 金属螯合剂的存在或不存在。将测量重组蛋白的核糖核酸酶活性 在锌配位残基中随着锌的石化和突变的变化。一系列带有氨基的蛋白质 酸变化可能会发现锌结合与酶促功能之间的联系，以建立 RNA裂解的反应机制。 RNA编辑的活性测定，包括添加外源性 将开发DYW - 脱氨酶。具有不同锌齿状分析的Dyw脱氨酶的活性将是 测量以确定锌离子结合与氨基水解酶活性之间的关系。第二个锌 离子在配体结合中可能具有专门的作用，并测试这种可能性的核酸配体要求 DYW - 脱氨酶的含量将使用带有修饰核苷酸的RNA和推定的底物确定。 最后，将筛选重组蛋白的结晶条件。将来可以使用晶体 旨在求解DYW脱氨酶结构的实验。