Structure function investigations of radical transfer and disulfide exchange in a class Ia ribonucleotide reductase

Ia类核糖核苷酸还原酶自由基转移和二硫键交换的结构功能研究

• 批准号: 10542661
• 负责人: Dana Westmoreland
• 金额: $ 6.95万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10542661
• 关键词: Active Sites; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Antiviral Agents; Area; Binding; Biochemical; Biological; Biological Assay; C-terminal; Catalysis; Chemicals; Chemistry; Competence; Complex; Coupled; Cryoelectron Microscopy; Crystallization; Crystallography; Cysteine; DNA Repair; DNA biosynthesis; Data; Deoxyribonucleotides; Development; Dissociation; Disulfides; Drug Targeting; Electron Transport; Electrons; Enzymes; Escherichia coli; Generations; Institution; International; Investigation; Knowledge; Label; Location; Maps; Mentorship; Methods; Monitor; Mutation; NADP; Oxidation-Reduction; Pathogenicity; Pathway interactions; Peptides; Pharmaceutical Preparations; Process; Proteins; Protons; Publications; Publishing; Reaction; Research; Resolution; Ribonucleotide Reductase; Ribonucleotide Reductase Subunit; Ribonucleotides; Role; Science; Side; Site; Site-Directed Mutagenesis; Spectrum Analysis; Structure; TXN gene; Tail; Techniques; Time; Training; Tryptophan; Tyrosine; Variant; Viral; Visit; Visualization; Water; Work; analog; anti-cancer; cofactor; collaborative environment; novel; prototype; skills; structural biology; thioredoxin reductase; water channel

PROJECT SUMMARY/ABSTRACT Class Ia ribonucleotide reductases (RNRs) are anti-cancer and antibiotic drug targets due to their role in the biosynthesis of deoxyribonucleotides. They convert ribonucleotides to deoxyribonucleotides using radical- based chemistry with reducing equivalents coming from a pair of cysteine residues in the active site that form a disulfide upon enzyme turnover. Recent structural work elucidating the active state structure of the class Ia RNR of Escherichia coli has revealed for the first time, the intact 32-Å-long pathway for radical transfer between subunits that is responsible for the generation of a catalytically essential radical species in the enzyme active site. In this proposed research, I will investigate the role of water channels in the proton transfer that occurs concomitant with electron transfer. I will also investigate the role of a tryptophan residue (W48) in a radical transfer pathway that otherwise involves tyrosine residues. Furthermore, I will probe the structural rearrangements that occur in order to facilitate re-reduction of the active site disulfide. This re-reduction pathway is known to be dependent on the C-terminal tail of the α subunit of RNR, which shuttles reducing equivalents from thioredoxin to the active site, but no structural snapshots of this process have ever been captured. Here, I propose to use site-specific mutagenesis to investigate the involvement of water channels in proton transfer by assessing the catalytic competency of RNR variants in which the water networks have been disrupted. I intend to assess the role of W48 in the PCET pathway through the site-specific incorporation of unnatural W analogues which will give us a spectroscopic handle with which to directly monitor radical transfer to and from W48. Finally, I propose to capture structural snapshots of the re-reduction steps of RNR, including a structure of RNR with thioredoxin using cryogenic electron microscopy. These studies will enable us to answer several of the major outstanding questions of this prototypical RNR. This work will also enable me, a physical inorganic and materials chemist by training, to gain expertise in biochemical and structural biology methods which will inform new research directions that will benefit from a synthesis of these two skill sets. As one of the world’s foremost research institutions, MIT is an ideal and highly collaborative environment in which to pursue the studies described in this proposal. Furthermore, as an internationally recognized leader in the field of structural biology, Prof. Drennan’s scientific mentorship will enable me to excel in my undertaking of this research.

项目概要/摘要 Ia 类核糖核苷酸还原酶 (RNR) 由于其在 脱氧核糖核苷酸的生物合成。他们使用自由基将核糖核苷酸转化为脱氧核糖核苷酸 基于化学的还原当量来自活性位点中的一对半胱氨酸残基，形成 酶周转时产生二硫键。最近的结构工作阐明了 Ia 类的活性态结构 大肠杆菌的 RNR 首次揭示了完整的 32 Å 长的自由基转移途径 亚基之间负责产生催化必需的自由基物种 酶的活性位点。在这项拟议的研究中，我将研究水通道在质子转移中的作用 这与电子转移同时发生。我还将研究色氨酸残基 (W48) 在 自由基转移途径，否则涉及酪氨酸残基。此外，我将探讨结构 为了促进活性位点二硫化物的重新还原而发生的重排。这次重新减持 已知该途径依赖于 RNR α 亚基的 C 末端尾部，该尾部穿梭减少 从硫氧还蛋白到活性位点的等价物，但尚未获得该过程的结构快照 被捕获。在这里，我建议使用位点特异性诱变来研究水通道在 通过评估 RNR 变体的催化能力来进行质子转移，其中水网络已被 扰乱了。我打算通过位点特异性掺入来评估 W48 在 PCET 途径中的作用 非自然的 W 类似物将为我们提供直接监测自由基转移的光谱手柄 往返 W48。最后，我建议捕获 RNR 重新降低步骤的结构快照，包括 使用低温电子显微镜观察 RNR 与硫氧还蛋白的结构。这些研究将使我们能够 回答这个原型 RNR 的几个主要悬而未决的问题。这项工作也将使我成为一个 物理无机和材料化学家通过培训，获得生物化学和结构生物学方面的专业知识 这些方法将为新的研究方向提供信息，这些方向将从这两种技能的综合中受益。作为 麻省理工学院是世界上最重要的研究机构之一，它是一个理想的、高度协作的环境， 进行本提案中描述的研究。此外，作为国际公认的领导者 在结构生物学领域，Drennan 教授的科学指导将使我能够在这一领域取得优异的成绩 研究。