Investigating the structural assembly of RNR multimers

研究 RNR 多聚体的结构组装

• 批准号: 8909392
• 负责人: Chris G Dealwis
• 金额: $ 0.8万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8909392
• 关键词: Affect; Allosteric Regulation; Allosteric Site; Antineoplastic Agents; Binding; Biochemical; Biological Assay; Catalysis; Catalytic Domain; Childhood Leukemia; Clofarabine; Complex; Cryoelectron Microscopy; Crystallization; Data; Data Reporting; Deoxyribonucleotides; Drug usage; Electron Microscopy; Enzymes; Equilibrium; Escherichia coli; Eukaryota; Free Radicals; Higher Order Chromatin Structure; Housing; Human; Laboratories; Lead; Life; Malignant neoplasm of pancreas; Maps; Mutation; Nucleotides; Outcome; Pharmaceutical Preparations; Physiological; Population; Preparation; RRM1 gene; Resolution; Ribonucleotide Reductase; Ribonucleotide Reductase Inhibitor; Ribonucleotides; Roentgen Rays; Saccharomyces cerevisiae; Scanning Transmission Electron Microscopy Procedures; Site-Directed Mutagenesis; Solutions; Structure; Sulfhydryl Compounds; X-Ray Crystallography; Yeasts; analytical ultracentrifugation; base; density; design; dimer; flexibility; gain of function; gemcitabine; improved; inhibitor/antagonist; insight; light scattering; mutant; particle; ribonucleotide reductase M2; stoichiometry; tool

DESCRIPTION (provided by applicant): Ribonucleotide reductase (RR) key to maintaining life as it catalyzes the slowest step in de novo DNTP synthesis by reducing ribonucleotides to deoxy ribonucleotides. RR is a multi-subunit enzyme consisting of a subunit that contains two allosteric sites and a catalytic site, and a b subunit that houses a free-radical that initiates thol-based catalysis. For almost 40 years RRs were thought to exist as heterotetramers. This organization failed, however, to explain how dATP inactivates and ATP activates the enzyme. Recently, this view has been challenged by biochemical data from other labs and structural data reported by us that support the existence of higher-order oligomers induced by its activator ATP and its inhibitor dATP. Interestingly, though ATP is an activator and dATP is an inhibitor of RR, they both cause the subunit to hexamerize. We propose that the two hexamers have different packing arrangements which lead to opposite outcomes of RR activity. Additionally, two important cancer drugs, gemcitabine and clofarabine are known to bind to higher-order RR oligomers. Thus, it is becoming increasingly clear that for an understanding of how RR is regulated in eukaryotes and targeted by cancer drugs, it is essential to elucidate the structure of higher-order oligomers formed by eukaryotic RRs. As higher-order holo-complexes may not be amenable to X-ray crystallography, we propose to use X-ray crystallography to determine the high-resolution structures of RR1 oligomers and single-particle electron microscopy (EM) to elucidate the organization of RR1 and RR2 in the holo-complexes.

描述（由申请人提供）：核糖核苷酸还原酶（RR）保持生命的键，因为它通过将核糖核苷酸还原为脱氧核糖核苷酸，从而催化了从头DNTP合成中最慢的步骤。 RR是一种由一个亚基组成的多生成酶，该酶包含两个变构位点和一个催化位点，以及一个容纳基于THOL的催化的自由基的B亚基。近40年来，RR被认为存在是异光聚体。但是，该组织未能解释DatP如何灭活和ATP激活酶。最近，通过来自其他实验室的生化数据和我们报道的结构数据的挑战，这些数据支持其激活剂ATP及其抑制剂DATP引起的高阶低聚物的存在。有趣的是，尽管ATP是一个激活剂，而DATP是RR的抑制剂，但它们都导致亚基进行六聚体。我们建议这两个六聚体具有不同的包装布置，从而导致RR活动的相反结果。此外，已知两种重要的癌症药物，吉西他滨和克洛法拉滨与高阶RR低聚物结合。因此，越来越清楚的是，要了解真核生物中RR的调节和癌症药物的靶向，必须阐明其结构 由真核RRS形成的高阶低聚物。由于较高的整体复合物可能不适合X射线晶体学，因此我们建议使用X射线晶体学来确定RR1低聚物和单粒子电子显微镜（EM）的高分辨率结构，以阐明Holo-complexes中RR1和RR2的组织。