Biomolecular Structure and Mechanism, Structure-Based Drug Design

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、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，代表了真核生物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，该结构揭示了衬底选择的两个标尺。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Drosha和Dicer的结构。然而，这些结构要么不含RNA，因此不能解释它们的作用机制，要么含有RNA，但不能解释为什么蛋白质是dsRNA特异性的。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Drosha和Dicer。Drosha与RNA的模型复合体解释了Drosha酶如何识别初级microRNA底物的基础连接处的最后一个碱基对，并测量11个核苷酸以确定剪切键在切割位点上的位置。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。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-7,8-二氢蝶呤焦磷酸激酶（HPPK，一种微生物必需的叶酸途径酶，但在哺乳动物中不存在）和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、酵母RNase III和人类DDX3X（一种DEAD-box解旋酶，以atp依赖的非过程方式解绕短RNA双链）的功能周期。这里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 have 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, 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, representing the first structure of a eukaryotic RNase III in complex with RNA in a catalytically meaningful manner. Strikingly, the structure reveals 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 Drosha and Dicer. These structures, however, either do not contain RNA and thus are not able to explain their mechanisms of action, or contain RNA but cannot explain why the proteins are dsRNA specific. 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 for both prokaryotic and eukaryotic enzymes, models with RNA can be reliably constructed for Drosha and Dicer. 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. 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. 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) and 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, yeast RNase III, and human DDX3X (a DEAD-box helicase that unwinds short RNA duplexes in an ATP-dependent, non-processive manner). Several biomolecular systems mentioned here are attractive molecular targets and structure-based drug development is an integral part of our research.