Biomolecular Structure and Mechanism, Structure-Based Drug Design

生物分子结构与机制、基于结构的药物设计

• 批准号: 10014349
• 负责人: XINHUA JI
• 金额: $ 219.98万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014349
• 关键词: Base Pairing; Binding; Biogenesis; Cell division; Complex; Couples; Crystallization; DNA-Directed RNA Polymerase; Dicer Enzyme; Diphosphotransferases; Double-Stranded RNA; Drug Design; Endoribonucleases; Enzymes; Evolution; Family; Family member; Folic Acid; Gene Expression Regulation; Goals; Guanosine Triphosphate Phosphohydrolases; Length; Mammals; Maps; Measures; MicroRNAs; Modeling; Molecular Structure; Molecular Target; Nucleotides; Positioning Attribute; Proteins; RNA; RNA Processing; RNA-Protein Interaction; Reaction; Reporting; Research; Ribonuclease III; Ribonucleases; Site; Structure; System; Yeasts; antimicrobial drug; base; cell growth; drug development; enzyme model; enzyme pathway; microorganism; novel anticancer drug; phosphodiester; structural biology; therapeutic development; transcription factor

Our research is focused on RNA-processing proteins and RNA polymerase (RNAP)-associated transcription factors. We pioneered the structural analysis of dsRNA in complex with ribonuclease III (RNase III) enzymes. RNase III represents a family of dsRNA-specific endoribonucleases required for RNA maturation and gene regulation. Prokaryotic RNase III and eukaryotic Rnt1p, Dcr1, Drosha, and Dicer are representative members of the family. Previously, we reported a total of eleven crystal structures of a bacterial RNase III in complex with dsRNA at various catalytic stages of the enzyme, including the first structure of a catalytically meaningful RNase III-RNA complex and the structure of a catalytic stage immediately after the cleavage of the phosphodiester bond. Recently, we determined the crystal structure of a post-cleavage complex of Rnt1p from yeast, the first structure of a eukaryotic RNase III in complex with RNA in a catalytically meaningful manner. Strikingly, the structure features two rulers for substrate selection. This double-ruler mechanism represents an example of the evolution of substrate selectivity and provides a framework for understanding the catalytic mechanism of eukaryotic RNase IIIs. The worldwide effort in structural analysis of other eukaryotic RNase III enzymes resulted in several important structures, including the structures of Dicer, Dcr1, and Drosha. These structures, however, do not contain RNA and thus are not able to explain their mechanisms of action. Our structures of RNase III:dsRNA complexes greatly enhanced the significance of these important structures. Based on the protein-RNA interactions revealed by our structures of both prokaryotic and eukaryotic enzymes, models with RNA can be reliably constructed for Dicer, Dcr1, and Drosha. A model complex of Dicer with RNA explains how Dicer enzymes recognize the 2-nucleotide 3' overhang of dsRNA substrate and measure 22 nucleotides up to position the scissile bond over the cleavage site. A model complex of Dcr1 with RNA explains how homodimers of non-canonical Dicer enzymes bind cooperatively along dsRNA substrate such that the distance between active centers in adjacent homodimers is the length of 22 nt. A model complex of Drosha with RNA explains how Drosha enzymes recognize the last base pair in the basal junction of the primary microRNA substrate and measure 11 nucleotides up to position the scissile bond over the cleavage site. Our structural and mechanistic studies of biomolecular systems aim to reveal their reaction coordinates or functional cycle. To date, we have described the reaction coordinates of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK, a folate pathway enzyme essential for microorganisms but absent in mammals), the functional cycle of Era (an essential GTPase that couples cell growth with cell division), RapA (a Swi2/Snf2 protein that recycles RNA polymerase), bacterial RNase III, and yeast RNase III. Several biomolecular systems mentioned above are attractive molecular targets and structure-based drug development is an integral part of our research.

我们的研究重点是RNA加工蛋白和RNA聚合酶（RNAP）相关转录因子。我们率先进行了核糖核酸酶III （RNase III）复合物的dsRNA结构分析。RNase III代表了RNA成熟和基因调控所需的dsrna特异性核糖核酸内切酶家族。原核RNase III和真核Rnt1p、Dcr1、Drosha和Dicer是该家族的代表成员。之前，我们报道了细菌RNase III与dsRNA在酶的不同催化阶段的共11个晶体结构，包括催化意义的RNase III- rna复合物的第一个结构和磷酸二酯键断裂后立即催化阶段的结构。最近，我们从酵母中确定了Rnt1p切割后复合体的晶体结构，这是真核生物RNase III与RNA以催化有意义的方式复合体的第一个结构。引人注目的是，该结构具有两个用于基材选择的尺子。这种双标尺机制代表了底物选择性进化的一个例子，并为理解真核生物RNase iii的催化机制提供了一个框架。世界范围内对其他真核生物RNase III酶的结构分析得到了一些重要的结构，包括Dicer、Dcr1和Drosha的结构。然而，这些结构不含RNA，因此无法解释它们的作用机制。我们的RNase III:dsRNA复合物的结构极大地增强了这些重要结构的意义。基于我们的原核和真核酶结构揭示的蛋白质-RNA相互作用，RNA模型可以可靠地构建Dicer， Dcr1和Drosha。Dicer与RNA的模型复合体解释了Dicer酶如何识别dsRNA底物的2-核苷酸3'悬垂，并测量22个核苷酸以定位剪切键在切割位点上的位置。Dcr1与RNA的模型复合体解释了非规范Dicer酶的同型二聚体如何沿着dsRNA底物协同结合，使得相邻同型二聚体的活性中心之间的距离为22 nt。Drosha与RNA的模型复合体解释了Drosha酶如何识别初级microRNA底物底部连接的最后一个碱基对，并测量11个核苷酸以定位剪切键在切割位点上的位置。我们对生物分子系统的结构和机理研究旨在揭示它们的反应坐标或功能周期。迄今为止，我们已经描述了6-羟甲基-7,8-二氢蝶呤磷酸激酶（HPPK，微生物必需的叶酸途径酶，但在哺乳动物中不存在）的反应配位，Era（一种必需的GTPase，将细胞生长与细胞分裂偶联），RapA（一种循环RNA聚合酶的Swi2/Snf2蛋白），细菌RNase III和酵母RNase III的功能循环。上面提到的几种生物分子系统是有吸引力的分子靶点，基于结构的药物开发是我们研究的一个组成部分。