Biomolecular Structure and Mechanism, Structure-Based Drug Design

我们一直在研究三种RNA加工蛋白：RNase III[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中两个相邻腺苷的二甲基化）和ERA（一种保守的GTPase，在细菌中将细胞分裂与细胞生长速率结合在一起，在哺乳动物中是一种潜在的肿瘤抑制因子）。之前，我们已经建立了RNase III执行dsRNA切割的分步机制，该机制可以外推到该家族的其他成员，包括Rnt1p、Drosha和Dicer。今年，我们在KsgA研究方面取得了重大进展，在ERA研究方面取得了突破性进展。在甲基转移酶中，KsgA及其催化的反应在进化过程中是保守的。然而，这种酶识别底物的细节仍然未知。我们已经确定了KsgA的结构，无配体形式，与RNA的复合物，以及与RNA和s -腺苷型同型半胱氨酸（SAH，辅助因子s -腺苷型甲硫氨酸的反应产物）的复合物，提供了KsgA-RNA和KsgA-SAH相互作用的第一个结构信息。此外，这些结构显示了RNA结合时发生的构象变化如何产生辅因子结合位点。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是柔性的，每个基序都表现出两种不同的构象。在RNA结合后，这两个基序稳定在其中一种构象中，这与SAH的结合是相容的。Motif X也在RNA结合时稳定，直接参与SAH的结合。</P> <P&gt；ERA由一个n端GTPase结构域和一个RNA结合的KH结构域组成，是必不可少的核糖体生物发生因子。它结合16S rRNA和30S核糖体亚基。然而，其rna结合位点、两个结构域之间的功能关系及其在核糖体生物发生中的作用尚不清楚。我们已经确定了ERA的两种晶体结构，一个具有GDP的二元配合物和一个具有gtp类似物的三元配合物，并在3&amp；#8242；16S rRNA末端。在三元复合物中，12个核苷酸中的前9个被蛋白质识别。我们发现GTP结合是ERA识别RNA的先决条件，RNA识别刺激其GTP水解活性。基于这些数据和其他数据，我们提出了ERA的功能周期，表明该蛋白作为16S rRNA加工和成熟的伴侣蛋白和30S核糖体亚基组装的检查点。AUCA序列在细菌、古细菌和真核生物中高度保守，而CCUCC，被称为anti-Shine-Dalgarno序列，仅在非真核生物中保守。因此，这些数据表明，通过识别AUCA，以及通过识别CCUCC来识别非真核ERA蛋白的高度保守的ERA功能的共同机制。我们在基于结构的药物开发方面的努力主要集中在两个系统，谷胱甘肽s -转移酶（GST）和6-羟甲基-7,8-二氢蛋白焦磷酸激酶（HPPK）。GST是一个解毒酶超家族，以GST-alpha、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。之前，我们基于结构的前药设计旨在释放gst -pi过表达癌细胞中的一氧化氮的细胞毒性水平，已经产生了PABA/NO，其在体外和体内都具有与顺铂相似的抗癌活性。今年，我们提出了对PABA/NO进行结构修饰，以提高稳定性、溶解度和同工酶特异性。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。哺乳动物从饮食中摄取叶酸，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对微生物是独特的，不是任何现有抗生素的靶点。因此，它是开发新型抗菌药物的理想靶点。基于结构的先导化合物的设计和表征正在进行中。&lt；

<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>