Biomolecular Structure and Mechanism, Structure-Based Drug Design

RNA干扰是由核糖核酸酶III （RNase III）家族成员（包括Dicer）产生的小干扰RNA介导的。对于机制研究，细菌RNase III已成为整个家族的有价值的模型系统。之前，我们已经展示了酶的内切酶结构域的二聚化如何在两个催化位点所在的地方创造一个催化谷，催化谷如何以一种方式容纳dsRNA，使两条RNA链中的每一条都与两个催化位点中的一个对齐，每条链的水解如何涉及两个亚基（一个亚基的残基参与了剪刀键的选择），而来自伴侣亚基的那些则参与了裂解化学)，以及RNase III如何利用这两个催化位点在其产物中产生2-核苷酸3'悬垂。最近，我们已经证明了Mg2+如何对催化能力强的蛋白质- rna复合物的形成至关重要，两个Mg2+离子如何驱动每个磷酸二酯键的水解，以及底物和蛋白质的构象变化如何成为组装催化复合物的关键因素。此外，我们以一种有意义的方式模拟了蛋白质-底物复合物和蛋白质-反应中间（过渡态）复合物。综上所述，模型和晶体结构提示了酶执行磷酰转移反应的逐步机制。细菌RNase III蛋白与dsRNA相互作用的结构信息和加工dsRNA的机制可以外推到其他家族成员，包括真核生物Rnt1p、Drosha和Dicer。RapA是一种具有atp酶活性的RNA聚合酶（RNAP）相关的Swi2/Snf2蛋白，在细胞中含量与sigma 70一样丰富。它在转录过程中刺激RNAP循环。最近，我们报道了RapA的第一个结构，这也是整个Swi2/Snf2家族的第一个全长结构。RapA包含七个结构域，其中两个表现出新的蛋白质折叠。RapA与ATP和双链DNA复合物的模型表明，RapA可能与dsDNA结合并在dsDNA上转位。我们的动态模板转换实验表明，RapA促进了转录后/终止后复合体（PTC）中隔离的RNAP的释放，从而进行转录再起始。我们的体外竞争实验表明，RapA仅与核心RNAP结合，但很容易被sigma 70取代。RapA可能是另一种通用转录因子，其结构为未来研究这种细菌Swi2/Snf2蛋白及其在转录过程中RNAP循环中的重要作用提供了框架。谷胱甘肽s -转移酶（GST）是一个解毒酶超家族，以GST- α、GST-mu、GST-pi等为代表。GST- α是人类肝脏中GST的主要亚型，对我们的健康起着重要的作用。GST-pi在许多形式的癌症中过度表达，从而为选择性靶向癌细胞提供了机会。我们基于结构的前药设计旨在在gst -pi过表达的癌细胞中释放细胞毒性水平的一氧化氮，产生PABA/NO，其在体外和体内均表现出与顺铂相似的抗癌活性。该设计基于基态和过渡态的GST结构。基态结构概述了不同同工酶的底物结合位点的形状和性质，过渡态结构信息为前药分子的结构修饰提供了指导。gst - α选择性化合物的两个关键改变导致了gst - α选择性PABA/NO。RNA干扰是由核糖核酸酶III （RNase III）家族成员（包括Dicer）产生的小干扰RNA介导的。对于机制研究，细菌RNase III已成为整个家族的有价值的模型系统。之前，我们已经展示了酶的内切酶结构域的二聚化如何在两个催化位点所在的地方创造一个催化谷，催化谷如何以一种方式容纳dsRNA，使两条RNA链中的每一条都与两个催化位点中的一个对齐，每条链的水解如何涉及两个亚基（一个亚基的残基参与了剪刀键的选择），而来自伴侣亚基的那些则参与了裂解化学)，以及RNase III如何利用这两个催化位点在其产物中产生2-核苷酸3'悬垂。最近，我们已经证明了Mg2+如何对催化能力强的蛋白质- rna复合物的形成至关重要，两个Mg2+离子如何驱动每个磷酸二酯键的水解，以及底物和蛋白质的构象变化如何成为组装催化复合物的关键因素。此外，我们以一种有意义的方式模拟了蛋白质-底物复合物和蛋白质-反应中间（过渡态）复合物。综上所述，模型和晶体结构提示了酶执行磷酰转移反应的逐步机制。细菌RNase III蛋白与dsRNA相互作用的结构信息和加工dsRNA的机制可以外推到其他家族成员，包括真核生物Rnt1p、Drosha和Dicer。RapA是一种具有atp酶活性的RNA聚合酶（RNAP）相关的Swi2/Snf2蛋白，在细胞中含量与sigma 70一样丰富。它在转录过程中刺激RNAP循环。最近，我们报道了RapA的第一个结构，这也是整个Swi2/Snf2家族的第一个全长结构。RapA包含七个结构域，其中两个表现出新的蛋白质折叠。RapA与ATP和双链DNA复合物的模型表明，RapA可能与dsDNA结合并在dsDNA上转位。我们的动态模板转换实验表明，RapA促进了转录后/终止后复合体（PTC）中隔离的RNAP的释放，从而进行转录再起始。我们的体外竞争实验表明，RapA仅与核心RNAP结合，但很容易被sigma 70取代。RapA可能是另一种通用转录因子，其结构为未来研究这种细菌Swi2/Snf2蛋白及其在转录过程中RNAP循环中的重要作用提供了框架。谷胱甘肽s -转移酶（GST）是一个解毒酶超家族，以GST- α、GST-mu、GST-pi等为代表。GST- α是人类肝脏中GST的主要亚型，对我们的健康起着重要的作用。GST-pi在许多形式的癌症中过度表达，从而为选择性靶向癌细胞提供了机会。我们基于结构的前药设计旨在在gst -pi过表达的癌细胞中释放细胞毒性水平的一氧化氮，产生PABA/NO，其在体外和体内均表现出与顺铂相似的抗癌活性。该设计基于基态和过渡态的GST结构。基态结构概述了不同同工酶的底物结合位点的形状和性质，过渡态结构信息为结构提供了指导[摘要截短为7800个字]。

RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family, including Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. Previously, we have shown how the dimerization of the endonuclease domain of the enzyme creates a catalytic valley where two catalytic sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two catalytic sites, how the hydrolysis of each strand involves both subunits (residues from one subunit are involved in the selection of the scissile bond, while those from the partner subunit are involved in the cleavage chemistry), and how RNase III uses the two catalytic sites to create the 2-nucleotide 3' overhangs in its products. Recently, we have demonstrated how Mg2+ is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modeled a protein-substrate complex and a protein-reaction intermediate (transition state) complex in a meaningful way. Together, the models and crystal structures suggest a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction. The structural information of protein-dsRNA interactions and the mechanism of dsRNA processing by bacterial RNase III can be extrapolated to other family members, including eukaryotic Rnt1p, Drosha and Dicer. RapA, as abundant as sigma 70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Recently, we reported the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription. Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design was based on GST structures at both ground state and transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state provided guidance for structural modifications of the prodrug molecules. Two key alterations of a GST-alpha-selective compound led to the GST-pi-selective PABA/NO.RNA interference is mediated by small interfering RNAs produced by members of the ribonuclease III (RNase III) family, including Dicer. For mechanistic studies, bacterial RNase III has been a valuable model system for the entire family. Previously, we have shown how the dimerization of the endonuclease domain of the enzyme creates a catalytic valley where two catalytic sites are located, how the catalytic valley accommodates a dsRNA in a manner such that each of the two RNA strands is aligned with one of the two catalytic sites, how the hydrolysis of each strand involves both subunits (residues from one subunit are involved in the selection of the scissile bond, while those from the partner subunit are involved in the cleavage chemistry), and how RNase III uses the two catalytic sites to create the 2-nucleotide 3' overhangs in its products. Recently, we have demonstrated how Mg2+ is essential for the formation of a catalytically competent protein-RNA complex, how the use of two Mg2+ ions can drive the hydrolysis of each phosphodiester bond, and how conformational changes in both the substrate and the protein are critical elements for assembling the catalytic complex. Moreover, we have modeled a protein-substrate complex and a protein-reaction intermediate (transition state) complex in a meaningful way. Together, the models and crystal structures suggest a stepwise mechanism for the enzyme to execute the phosphoryl transfer reaction. The structural information of protein-dsRNA interactions and the mechanism of dsRNA processing by bacterial RNase III can be extrapolated to other family members, including eukaryotic Rnt1p, Drosha and Dicer. RapA, as abundant as sigma 70 in the cell, is an RNA polymerase (RNAP)-associated Swi2/Snf2 protein with ATPase activity. It stimulates RNAP recycling during transcription. Recently, we reported the first structure of RapA, which is also the first full-length structure for the entire Swi2/Snf2 family. RapA contains seven domains, two of which exhibit novel protein folds. Our model of RapA in complex with ATP and double-stranded (ds) DNA suggests that RapA may bind to and translocate on dsDNA. Our kinetic template-switching assay shows that RapA facilitates the release of sequestered RNAP from a posttranscrption/posttermination complex (PTC) for transcription reinitiation. Our in vitro competition experiment indicates that RapA binds to core RNAP only but is readily displaceable by sigma 70. RapA is likely another general transcription factor, the structure of which provides a framework for future studies of this bacterial Swi2/Snf2 protein and its important roles in RNAP recycling during transcription. Glutathione S-transferase (GST) is a superfamily of detoxification enzymes, represented by GST-alpha, GST-mu, GST-pi, etc. GST-alpha is the predominant isoform of GST in human liver, playing important roles for our well being. GST-pi is overexpressed in many forms of cancer, thus presenting an opportunity for selective targeting of cancer cells. Our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells yielded PABA/NO, which exhibited anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. The design was based on GST structures at both ground state and transition state. The ground-state structures outlined the shape and property of the substrate-binding site in different isozymes, and the structural information at the transition-state provided guidance for structur [summary truncated at 7800 characters]