Investigating the structural assembly of RNR multimers

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，因为它催化dNTP从头合成的最慢步骤，通过将核苷酸还原为脱氧核苷酸来实现。RR是一种多亚单位酶，由一个含有两个变构中心和一个催化中心的亚基和一个含有引发醇基催化的自由基的b亚基组成。近40年来，RRS一直被认为是以异构体的形式存在的。然而，这个组织未能解释dATP是如何使酶失活的，而ATP又是如何激活酶的。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道的结构数据的挑战，这些数据支持它的激活剂ATP和它的抑制剂dATP诱导的高阶低聚物的存在。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角化。我们认为这两个六角体具有不同的包装排列，这导致了RR活性的相反结果。此外，已知两种重要的抗癌药物吉西他滨和氯法拉滨可与高阶RR寡聚体结合。因此，越来越清楚的是，要了解RR在真核细胞中是如何调节的并被抗癌药物靶向，阐明其结构是至关重要的。 真核RRS形成的高阶低聚物。由于高阶全息络合物可能不符合X射线结晶学的要求，我们建议使用X射线结晶学来确定RR1齐聚物的高分辨结构，并用单粒子电子显微镜(EM)来阐明RR1和RR2在全息络合物中的组织。

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.