Structural Studies of Ribonucleotide Reductase Complexes

描述（由申请人提供）：核糖核苷酸还原酶（RNRs）是一种多亚基酶，是抗癌治疗的重要靶点，因此与人类健康直接相关。完整的la类RNRs复合物，包括人类和大肠杆菌的RNRs，从未通过结构方法可视化过。此外，被抑制的Ia类RNR复合物的寡聚状态还没有很好地确定，有人认为被抑制的RNR可能以比活性复合物更高阶的低聚物存在。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，已被证明可以产生稳定的抑制人类和大肠杆菌rrna的复合物。本文采用x射线晶体学和小角x射线散射（SAXS）技术对大肠杆菌和人类RNRs进行了结构研究。目的1：用SAXS法测定人和大肠杆菌rrna的四级结构。SAXS结构可用于验证对接模型和未来RNR配合物的晶体结构。所提出的解决方案SAXS研究将深入了解大肠杆菌和人类RNRs如何在抑制剂的作用下形成不同的低聚物种，更重要的是，四级结构如何调节la类RNRs的整体活性。目的2：测定吉西他滨抑制人RNR的晶体结构。吉西他滨结合的人类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。这种结构也将使我们能够首次观察亚基之间的结合相互作用，并为未来设计靶向这种酶的化疗药物提供见解。

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.