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: 10386378
• 负责人: Dana Westmoreland
• 金额: $ 6.72万
• 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/10386378
• 关键词: Active Sites; Amino Acids; Anabolism; Antibiotics; Antineoplastic Agents; Antiviral Agents; Area; Biochemical; Biological; Biological Assay; C-terminal; Catalysis; Chemicals; Chemistry; Competence; Complex; Coupled; Crystallization; Crystallography; Cysteine; DNA Repair; DNA biosynthesis; Data; Deoxyribonucleotides; Development; Dissociation; Disulfides; Drug Targeting; Electron Microscopy; Electron Transport; Electrons; Enzymes; Escherichia coli; Generations; Institution; International; Investigation; Iron; 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; Water; Work; analog; anti-cancer; antineoplastic antibiotics; base; cofactor; collaborative environment; cryogenics; novel; 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.

项目摘要/摘要 Ia类核糖核苷酸还原酶(RNRs)是抗癌和抗生素药物的靶点，因为它们在 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- 基础化学，还原等价物来自活性中心的一对半胱氨酸残基，形成 二硫化物对酶的周转。最近的结构工作阐明了Ia类的活性状态结构 大肠埃希氏菌RnR首次揭示了完整的32？长自由基转移途径 在亚基之间负责产生催化必需的自由基物种 酶活性部位。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。 这是伴随着电子转移而发生的。我还将调查色氨酸残基(W48)在 自由基转移途径，否则涉及酪氨酸残基。此外，我将探讨结构上的 为了促进活性中心二硫化物的重新还原而发生的重排。这一重新减少 已知的途径依赖于RNR亚单位的α亚单位的C-末端尾巴，它穿梭于减少 从硫氧还蛋白到活性部位的等价物，但这一过程的结构快照从未被 被抓了。在这里，我建议使用定点突变来研究水通道在 通过评估水网络中RNR变体的催化能力来实现质子转移 打乱了。我打算通过位点特异性的掺入来评估W48在PCET途径中的作用 非天然的W类似物，这将给我们一个直接监测自由基转移的光谱手柄 往返48号州际公路。最后，我建议捕获RNR再减少步骤的结构快照，包括 用低温电子显微镜观察硫氧还蛋白与RNR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，一个 物理无机和材料化学家，通过培训获得生化和结构生物学方面的专业知识 方法将为新的研究方向提供信息，这些新的研究方向将受益于这两种技能的综合。AS 作为世界上最顶尖的研究机构之一，麻省理工学院是一个理想的高度协作的环境 继续进行本提案中所述的研究。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出色地完成这项工作 研究。