Biomolecular Structure and Mechanism, Structure-Based Drug Design

我们的研究主要集中在RNA加工蛋白和RNA聚合酶(RNAP)相关的转录因子上。我们开创了dsRNA与核糖核酸酶III(RNaseIII)酶复合体的结构分析。RNaseIII代表RNA成熟和基因调控所需的dsRNA特异性内切核酸酶家族。原核生物RNaseIII和真核生物Rnt1p、Dcr1、DROSHA和DICER是该家族的代表性成员。此前，我们报道了细菌RNaseIII与dsRNA的复合体在酶的不同催化阶段的11种晶体结构，包括具有催化意义的RNaseIII-RNA复合体的第一结构和磷酸二酯键断裂后立即催化阶段的结构。最近，我们确定了酵母Rnt1p裂解后复合体的晶体结构，这是真核RNaseIII与RNA以催化有意义的方式形成的复合体的第一个结构。引人注目的是，该结构具有两个尺子用于基板选择。这一双标尺机制代表了底物选择性进化的一个例子，并为理解真核细胞RNaseIII的催化机制提供了一个框架。世界范围内对其他真核生物RNaseIII酶的结构分析工作导致了几个重要的结构，包括DICER、Dcr1和DROSHA的结构。然而，这些结构不包含RNA，因此无法解释它们的作用机制。我们的RNaseIII：dsRNA复合体的结构极大地增强了这些重要结构的意义。基于我们的原核生物和真核生物酶的结构所揭示的蛋白质-RNA相互作用，可以可靠地为DICER、DCR1和DROSHA构建带有RNA的模型。Dester与RNA的模型复合体解释了Diller酶如何识别dsRNA底物的2-核苷酸3‘悬垂，并测量22个核苷酸以将剪切键定位在切割位点上。Dcr1与RNA的模型络合物解释了非规范DICER酶的同源二聚体是如何沿着dsRNA底物协同结合的，从而使相邻同源二聚体中的活性中心之间的距离为22nT。DROSHA与RNA的模型复合体解释了DROSHA酶如何识别初级microRNA底物基底部的最后一个碱基对，并测量11个核苷酸以将剪切键定位在切割位点上。我们对生物分子系统的结构和机理的研究旨在揭示它们的反应坐标或功能循环。到目前为止，我们已经描述了6-羟甲基-7，8-二氢蝶呤焦磷酸激酶(HPPK，一种对微生物必不可少但在哺乳动物中不存在的叶酸途径酶)、Era(一种将细胞生长与细胞分裂结合在一起的必需GTP酶)、RapA(一种回收RNA聚合酶的Swi2/Snf2蛋白)、细菌RNaseIII和酵母RNaseIII的反应坐标。上述几个生物分子系统是吸引人的分子靶标，基于结构的药物开发是我们研究的一个组成部分。

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.