Biomolecular Structure and Mechanism, Structure-Based Drug Design

&lt；P&gt；我们一直在研究三种RNA加工蛋白：RNaseIII[启动RNA干扰的双链RNA特异性内切酶家族]，KsgA(一种普遍保守的甲基转移酶，催化小亚基核糖体RNA中相邻两个腺苷的二甲基化)和ERA(一种保守的GTP酶，在细菌中将细胞分裂与细胞生长速度结合在一起，在哺乳动物中是一种潜在的肿瘤抑制因子)。此前，我们已经为RNaseIII建立了一种逐步执行dsRNA切割的机制，这可以外推到该家族的其他成员，包括Rnt1p、DROSHA和Disher。今年，我们在KsgA研究和ERA研究方面取得了重大进展。在甲基转移酶中，KsgA及其催化的反应在整个进化过程中都是保守的。然而，该酶识别底物的具体情况仍不清楚。我们测定了KsgA的结构，包括无配体形式、与核糖核酸的络合物以及与核糖核酸和S-腺苷同型半胱氨酸的络合物(SAH，辅因子S-腺苷蛋氨酸的反应产物)，提供了有关KsgA-RNA和KsgA-SAH相互作用的第一批结构信息。此外，这些结构显示了RNA结合时发生的构象变化如何创建辅因子结合位点。KsgA中有9个保守的功能基序(基序I-VIII和X)。在RNA结合之前，基序I和VIII是灵活的，每个基序都表现出两种不同的构象。当RNA结合时，两个基序在其中一种构象中变得稳定，这与SAH的结合是相容的。基序X也稳定在RNA结合上，直接参与SAH的结合。&lt；/P&gt；&P&gt；ERA由一个N末端的GTP酶结构域和一个RNA结合的KH结构域组成，是一个重要的核糖体生物发生因子。它与16S rRNA和30S核糖体亚基结合。然而，它的RNA结合部位、两个结构域之间的功能关系以及它在核糖体生物发生中的作用仍不清楚。我们测定了ERA的两个晶体结构，一个是与GDP的二元配合物，一个是与GTP类似物的三元配合物，以及16S rRNA 3&amp；末端的1531AUCACCUCCUUA1542序列。在三元复合体中，12个核苷酸中的前9个由蛋白质识别。我们证明GTP结合是ERA识别RNA的先决条件，RNA识别刺激其GTP水解性。基于这些和其他数据，我们提出了ERA的功能循环，认为该蛋白作为16S rRNA加工和成熟的伴侣和30S核糖体亚单位组装的检查点。AucA序列在细菌、古菌和真核生物中高度保守，而CCUCC，即反Shine-Dalgarno序列，只在非真核生物中保守。因此，这些数据表明，通过识别AucA，在所有三个生命王国中都有一个高度保守的ERA功能的共同机制，而对于非真核ERA蛋白，通过识别CCUCC；/P&gt；&P&gt；，我们在基于结构的药物开发方面的努力一直集中在两个系统上，谷胱甘肽S转移酶(Gst)和6-羟甲基-7，8-二氢磷酸脱氢酶(HPPK)。GST是一个解毒酶超家族，以GST-α、GST-Mu、GST-pi等为代表。GST-α是人体肝脏中主要的GST亚型，对人类的健康起着重要的作用。GST-pi在许多形式的癌症中过表达，因此为选择性靶向癌细胞提供了机会。此前，我们的基于结构的前药设计旨在释放GST-pi过表达癌细胞中细胞毒水平的一氧化氮，已经产生了PABA/NO，它在体外和体内都表现出抗癌活性，其效力类似于顺铂。今年，我们建议对PABA/NO进行结构修饰，以提高稳定性、溶解性和同工酶特异性。HPPK是叶酸生物合成途径中的关键酶。叶酸辅助因子是生命所必需的。哺乳动物从饮食中获得叶酸，而大多数微生物必须从头合成叶酸。因此，叶酸途径是开发抗菌药物的理想靶点。此外，HPPK对微生物来说是独一无二的，并不是任何现有抗生素的靶标。因此，它是开发新型抗菌剂的理想靶标。基于结构的先导化合物设计和表征正在进行中。

<P>We have been studying three RNA-processing proteins: RNase III [a family of double-stranded (ds) RNA-specific endonucleases that initiate RNA interference], KsgA (a universally conserved methyltransferase that catalyzes the dimethylation of two adjacent adenosines in small-subunit ribosomal RNA), and ERA (a conserved GTPase that couples cell division with cell growth rate in bacteria and is a potential tumor suppressor in mammals). Previously, we have established a stepwise mechanism for RNase III to execute dsRNA cleavage, which can be extrapolated to other members of the family, including Rnt1p, Drosha and Dicer. This year, we have made significant progress in KsgA research and breakthrough advances in ERA research.</P> <P>Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. We have determined structures of KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), providing the first pieces of structural information on KsgA-RNA and KsgA-SAH interactions. Moreover, the structures show how conformational changes that occur upon RNA binding create the cofactor-binding site. There are nine conserved functional motifs (motifs I-VIII and X) in KsgA. Prior to RNA binding, motifs I and VIII are flexible, each exhibiting two distinct conformations. Upon RNA binding, the two motifs become stabilized in one of these conformations, which is compatible with the binding of SAH. Motif X, which is also stabilized upon RNA binding, is directly involved in the binding of SAH.</P> <P>ERA, composed of an N-terminal GTPase domain followed by an RNA-binding KH domain, is an essential ribosome biogenesis factor. It binds to 16S rRNA and the 30S ribosomal subunit. However, its RNA-binding site, the functional relationship between the two domains, and its role in ribosome biogenesis remain unclear. We have determined two crystal structures of ERA, a binary complex with GDP and a ternary complex with a GTP-analog and the 1531AUCACCUCCUUA1542 sequence at the 3′ end of 16S rRNA. In the ternary complex, the first nine of the 12 nucleotides are recognized by the protein. We show that GTP binding is a prerequisite for RNA recognition by ERA and that RNA recognition stimulates its GTP-hydrolyzing activity. Based on these and other data, we propose a functional cycle of ERA, suggesting that the protein serves as a chaperone for processing and maturation of 16S rRNA and a checkpoint for assembly of the 30S ribosomal subunit. The AUCA sequence is highly conserved among bacteria, archaea, and eukaryotes, whereas the CCUCC, known as the anti-Shine-Dalgarno sequence, is conserved in non-eukaryotes only. Therefore, these data suggest a common mechanism for a highly conserved ERA function in all three kingdoms of life by recognizing the AUCA, with a twist for non-eukaryotic ERA proteins by also recognizing the CCUCC.</P> <P>Our effort in structure-based drug development has been focused on two systems, Glutathione S-transferase (GST) and 6-hydroxymethyl-7,8-dihydroptein pyrophosphokinase (HPPK). GST represents 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. Previously, our structure-based design of prodrugs intended to release cytotoxic levels of nitric oxide in GST-pi-overexpressing cancer cells had yielded PABA/NO, which exhibits anticancer activity both in vitro and in vivo with a potency similar to that of cisplatin. This year, we have proposed structural modifications to PABA/NO for improved stability, solubility, and isozyme specificity. HPPK is a key enzyme in the folate biosynthetic pathway. Folate cofactors are essential for life. Mammals derive folates from their diet, whereas most microorganisms must synthesize folates de novo. Thus, the folate pathway is an ideal target for developing antimicrobial agents. In addition, HPPK is unique for microorganisms and is not the target for any existing antibiotics. Therefore, it is an ideal target for developing novel antimicrobial agents. Structure-based design and characterization of lead compounds are in progress.</P>