Investigating the structural assembly of RNR multimers

研究 RNR 多聚体的结构组装

• 批准号: 8475488
• 负责人: Chris G Dealwis
• 金额: $ 28.35万
• 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/8475488
• 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）是维持生命的关键，因为它通过将核糖核苷酸还原为脱氧核糖核苷酸来催化DNTP从头合成中最慢的步骤。 RR 是一种多亚基酶，由一个包含两个变构位点和一个催化位点的亚基和一个包含可启动基于硫醇的催化作用的自由基的 b 亚基组成。近 40 年来，人们一直认为 RRs 以异四聚体的形式存在。然而，该组织未能解释 dATP 如何失活以及 ATP 如何激活该酶。最近，这一观点受到了来自其他实验室的生化数据和我们报告的结构数据的挑战，这些数据支持由其激活剂 ATP 及其抑制剂 dATP 诱导的高阶寡聚物的存在。有趣的是，虽然 ATP 是 RR 的激活剂，dATP 是 RR 的抑制剂，但它们都会导致亚基六聚化。我们认为这两个六聚体具有不同的堆积排列，从而导致 RR 活性的相反结果。此外，已知两种重要的癌症药物吉西他滨和氯法拉滨可与高阶 RR 寡聚物结合。因此，越来越清楚的是，为了了解 RR 在真核生物中如何调节以及如何被癌症药物靶向，有必要阐明 RR 的结构。 由真核生物 RR 形成的高阶寡聚物。由于高阶全息复合物可能不适合 X 射线晶体学，我们建议使用 X 射线晶体学来确定 RR1 寡聚物的高分辨率结构，并使用单粒子电子显微镜 (EM) 来阐明 RR1 的组织和 RR2 在全息复合体中。