Structural Studies of Ribonucleotide Reductase Complexes

描述（由申请人提供）：核糖核苷酸还原酶（RNR）是多亚基酶，是抗癌治疗的重要靶标，因此与人类健康直接相关。完整的la类RNR复合物，包括人和E.大肠杆菌RNR，从来没有被可视化的结构方法。此外，受抑制的Ia类RNR复合物的寡聚化状态没有很好地建立，并且已经表明受抑制的RNR可能作为比活性复合物更高级的寡聚体存在。与基于机制的抑制剂和化疗药物吉西他滨（2 '，2'-二氟-2 '-脱氧胞苷）二磷酸一起孵育，已显示产生稳定的人和大肠杆菌的抑制复合物。coli RNR。本文对E.大肠杆菌和人类RNR使用X射线晶体学和小角X射线散射（SAXS）。目的1：确定人和大肠杆菌的四级结构. coliRNR的溶液中的SAXS。小角X射线衍射结构可以用来验证对接模型和未来的RNR复合物的晶体结构。提出的解决方案SAXS研究将提供深入了解如何E。大肠杆菌和人RNR可以响应于抑制剂形成不同的寡聚物种类，更重要的是，四级结构如何调节Ia类RNR的总体活性。目的2：确定吉西他滨抑制人RNR的晶体结构。吉西他滨结合的人RNR复合物的晶体结构将使我们能够确定这种药物如何修饰活性位点并破坏催化必需的自由基转移。这种结构还将使我们能够首次观察亚基之间的结合相互作用，并为靶向这种酶的化疗药物的未来设计提供见解。 公共卫生相关性：核糖核苷酸还原酶是一种在所有生物体中发现的蛋白质，是吉西他滨等抗癌药物的重要靶点。然而，与许多其他作为药物靶点的蛋白质不同，这种蛋白质的完整结构从未得到解决。通过研究这种蛋白质的结构，我们将更多地了解目前正在使用的癌症药物，并有助于未来的药物设计工作。

DESCRIPTION (provided by applicant): Ribonucleotide reductases (RNRs) are multi-subunit enzymes that are important targets for anti-cancer therapy and thus, directly relevant to human health. Intact complexes of class la RNRs, which include human and E. coli RNRs, have never been visualized by structural methods. Additionally, the oligomerization states of the inhibited class Ia RNR complexes are not well established, and it has been suggested that the inhibited RNRs may exist as higher order oligomers than the active complex. Incubation with the mechanism-based inhibitor and chemotherapeutic drug, gemcitabine (2',2'-difluoro-2'-deoxycytidine) diphosphate, has been shown to yield stable inhibited complexes of human and E. coli RNRs. Here, we propose a structural study of E. coli and human RNRs using X-ray crystallography and small-angle X-ray scattering (SAXS). Aim 1: To determine the quaternary structures of human and E. coli RNRs in solution using SAXS. The SAXS structures can be used to validate the docking model and future crystal structures of RNR complexes. The proposed solution SAXS studies will provide insight into how E. coli and human RNRs may form different oligomeric species in response to inhibitors and more significantly, how quaternary structure may regulate the overall activity of class la RNRs. Aim 2: To determine the crystal structure of human RNR inhibited by gemcitabine. Crystal structures of the gemcitabine-bound human RNR complex will allow us to identify how this drug may modify the active site and disrupt the catalytically essential radical transfer This structure will also allow us to observe the binding interactions between the subunits for the first time as well as provide insight into the future design of chemotherapeutic drugs that target this enzyme. PUBLIC HEALTH RELEVANCE: Ribonucleotide reductase is a protein found in all organisms that is an important target for cancer drugs such as gemcitabine. Unlike many other proteins that are drug targets, however, the complete structure of this protein has never been solved. By studying the structure of this protein, we will understand more about cancer drugs that are currently in use and aid future drug design efforts.