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。我们开创了与核糖核酸酶III（RNase III）酶复合的dsRNA的结构分析。RNase III代表RNA成熟和基因调控所需的dsRNA特异性内切核糖核酸酶家族。蛋白质核糖核酸酶III和真核生物Rnt 1 p、Dcr 1、Drosha和Dicer是该家族的代表性成员。在此之前，我们报道了总共11个晶体结构的细菌核糖核酸酶III与双链RNA复合物在不同的催化阶段的酶，包括第一个结构的催化意义的核糖核酸酶III-RNA复合物和结构的催化阶段后立即裂解的磷酸二酯键。最近，我们确定了来自酵母的Rnt 1 p的裂解后复合物的晶体结构，这是真核RNA酶III以催化有意义的方式与RNA复合的第一种结构。引人注目的是，该结构具有用于基板选择的两个标尺。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RNA酶III的催化机制提供了一个框架。在其他真核生物RNase III酶的结构分析中，世界范围内的努力产生了几个重要的结构，包括Dicer，Dcr 1和Drosha的结构。然而，这些结构不含RNA，因此无法解释其作用机制。我们的RNase III：dsRNA复合物的结构大大增强了这些重要结构的意义。基于我们的原核和真核酶的结构所揭示的蛋白质-RNA相互作用，可以可靠地构建Dicer，Dcr 1和Drosha的RNA模型。Dicer与RNA的模型复合物解释了Dicer酶如何识别dsRNA底物的2个核苷酸的3'突出端，并测量22个核苷酸，直到将易断裂的键定位在切割位点上。Dcr 1与RNA的模型复合物解释了非典型Dicer酶的同源二聚体如何协同地沿沿着dsRNA底物结合，使得相邻同源二聚体中活性中心之间的距离为22 nt。Drosha与RNA的模型复合物解释了Drosha酶如何识别主要microRNA底物的基底连接中的最后一个碱基对，并测量11个核苷酸，以将易断裂的键定位在切割位点上。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，微生物必需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偶联的必需GT3），RapA（一种抑制RNA聚合酶的Swi 2/Snf 2蛋白），细菌RNase III和酵母RNase III的功能循环的反应坐标。上面提到的几个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。

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.