Structural Studies of Ribonucleotide Reductase Complexes

描述(申请人提供)：核糖核苷酸还原酶(RNRs)是多亚单位酶，是抗癌治疗的重要靶点，因此与人类健康直接相关。完整的1a RNRs类复合体，包括人和大肠杆菌RNRs，从未用结构方法可视化过。此外，被抑制的Ia类RNR络合物的齐聚状态还没有得到很好的确定，有人认为被抑制的RNR可能是以比活性络合物更高阶齐聚物的形式存在。与基于机理的抑制剂和化疗药物吉西他滨(2‘，2’-二氟-2‘-脱氧胞苷)二磷酸孵育，已被证明产生稳定的抑制人和大肠杆菌RNRs的复合体。在这里，我们提出了一种利用X射线结晶学和小角X射线散射(SAXS)对大肠杆菌和人类RNRs进行结构研究的方法。目的1：用SAXS法测定人和大肠杆菌RNRs在溶液中的四级结构。SAXS结构可用于验证RNR络合物的对接模型和未来的晶体结构。拟议的解决方案SAXS研究将深入了解大肠杆菌和人类RNRs如何形成不同的寡聚物种以响应抑制剂，更重要的是，四级结构如何调节1a类RNRs的整体活性。目的：测定吉西他滨抑制的人RNR的晶体结构。吉西他滨结合的人RNR复合体的晶体结构将使我们能够确定这种药物如何修饰活性部位并破坏催化必需的自由基转移。这种结构也将使我们能够首次观察亚基之间的结合作用，并为未来针对这种酶的化疗药物的设计提供洞察力。 与公共卫生相关：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被解决。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抗癌药物，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.