Structural and mechanistic studies of RNase H and HIV reverse transcriptase

RNase H 和 HIV 逆转录酶的结构和机制研究

• 批准号: 7734094
• 负责人: Wei Yang
• 金额: $ 34.78万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734094
• 关键词: Active Sites; Anti-HIV Agents; Bacteria; Binding; Biochemical; Catalytic Domain; Cells; Cellular Structures; Collaborations; Complex; Crystallization; DNA biosynthesis; Double-Stranded RNA; Embryo; Endoribonucleases; Enzymes; Excision; Failure; Funding; Genes; Genomics; Goals; HIV; HIV Infections; HIV Integrase; Human; Human Genome; Hybrids; Immune response; Ions; Knock-out; Length; Metals; Mitochondria; Modeling; Molecular; Mus; N-terminal; National Institute of Child Health and Human Development; Nucleic Acids; Numbers; Pancreatic ribonuclease; Play; Polymerase; Publishing; RNA; RNA primers; RNA-Directed DNA Polymerase; Range; Reverse Transcription; Ribonuclease H; Ribonucleases; Role; Specificity; Structure; Substrate Specificity; Variant; Viral; Virus; Virus Diseases; Writing; X-Ray Crystallography; analog; design; enzyme substrate complex; human ribonuclease H1; inhibitor/antagonist; insight; mutant; preference; recombinase; tool

RNase H (Ribonuclease H) is conserved in species ranging from bacteria to humans. It degrades the RNA strand in an RNADNA hybrid and removes RNA primers during DNA replication. Knock-out of rnh1 gene in mice results in embryonic lethality due to failure of mitochondrial DNA replication. HIV encodes its own viral RNase H activity, which is essential for converting genomic RNA to dsDNA before integration into human genome. In addition to removal of RNA primers during both (-) and (+) strand synthesis, HIV RNase H degrades viral genomic RNA after reverse transcription of the (-) strand and generates a primer for the (+) strand synthesis. As one of only four enzymes encoded by HIV and essential for its infection, the viral RNase H is an obvious target for developing anti-HIV drugs. A number of RNase H structures from cellular and viral origins have been determined by X-ray crystallography or NMR in the past 17 years, but an enzyme-substrate complex revealing how an RNA/DNA hybrid is recognized by RNase H remained elusive until two years ago. With the IATAP funding and in collaboration with Dr. Robert Crouchs group in NICHD, my group determined the first crystal structure of RNase H (from B. haloduran) complexed with an RNA/DNA hybrid substrate and elucidated the substrate specificity and catalytic mechanism in 2005. Our results were published in Cell. Since then, we have determined the crystal structures of B. haloduran RNase H bound to cleavage intermdiate analog and product, thereby resolving a long standing mystery about the requirement for two metal ions for nucleic acid synthesis and degradation. These results have been published in EMBO J. and Molecular Cell in 2005-2006. In the last fiscal year, we determined the crystal structure of human RNase H catalytic domain (RNase HC) complexed with RNA/DNA substrate to reveal the difference between cellular and viral enzymes. They share a conserved active site, but differ in substrate binding and cleavage specificity. Modeling of HIV reverse transcriptase (RT), which contains both the polymerase and RNase H activity, suggests that an RNA/DNA substrate cannot simultaneously occupy the polymerase and RNase H active sites and must undergo a conformational change to toggle between the two catalytic centers. The RT region that accommodates this conformational change offers a new target to develop HIV-specific inhibitors. The results have been written up and are in press at Molecular Cell. In this fiscal year, we have determined the N-terminal substrate binding domain of human RNase H in complex with RNA/DNA hybrid and characterized its binding preference of RNA/DNA hybrid over dsRNA and dsDNA. The results has been published in EMBO J. To capture a crystal structure of viral RNase H complexed with its substrate, we have produced the full-length HIV reverse transcriptase and RNase H catalytic mutant. Special RNA/DNA substrates are being designed to target the RNase active site for crystallization trials with HIV RT. In collaboration with Dr. Stuart Le Grice's group at NCI, we have started a broad screen of HIV RT variants and RNA/DNA hybrids for co-crystals with RNA/DNA substrate at the RNase H active site. References Nowotny, M., Gaidamakov, S. A., Ghirlando, R., Cerritelli, S. M., Crouch, R. J. and Yang, W. (2007). Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol. Cell, 28, 264-276.

RNase H（核糖核酸酶H）在从细菌到人类的物种中保守。 它在RNADNA杂交中降解RNA链，并在DNA复制过程中除去RNA底漆。小鼠中RNH1基因的敲除导致因线粒体DNA复制失败而导致胚胎致死性。 HIV编码其自身的病毒RNase H活性，这对于将基因组RNA转换为DSDNA至关重要，然后再整合到人类基因组中。 除了在（ - ）和（（+）链合成过程中除去RNA底漆外，HIV RNase H在（ - ）链逆转录后降解病毒基因组RNA，并为（+）链合成生成引物。 作为由HIV编码的四种酶之一，对其感染至关重要，病毒RNase H是开发抗HIV药物的明显靶标。在过去的17年中，已经通过X射线晶体学或NMR确定了许多来自细胞和病毒起源的RNase H结构，但是直到两年前，RNase H仍难以捉摸，揭示了RNA/DNA杂种如何识别出RNA/DNA杂交的酶。通过IATAP资金并与NICHD的Robert Crouchs Group合作，我的小组确定了与RNA/DNA杂交底物复合的RNase H（来自B. haloduran）的第一个晶体结构，并阐明了2005年的底物特异性和催化机制。我们的结果在细胞中发表。从那时起，我们确定了与裂解与裂解相互裂解类似物和产物结合的haloduran rNase H的晶体结构，从而解决了对两个金属离子对核酸合成和降解的需求的长期神秘。这些结果已在2005 - 2006年发表在Embo J.和Molecular细胞中。 在上一个财政年度，我们确定了与RNA/DNA底物复合的人RNase H催化结构域（RNase HC）的晶体结构，以揭示细胞和病毒酶之间的差异。它们共享一个保守的活性位点，但底物结合和切割特异性有所不同。同时包含聚合酶和RNase H活性的HIV逆转录酶（RT）的建模表明，RNA/DNA底物不能同时占据聚合酶和RNase H活性位点，并且必须经过两个催化中心之间切换的构象变化。适应这种构象变化的RT区域为开发HIV特异性抑制剂提供了一个新的目标。结果已写入，并在分子细胞处印刷。 在这个财政年度，我们已经确定了与RNA/DNA杂交的复合物中人RNase H的N末端底物结合结构域，并表征了其对RNA/DNA杂交的结合偏好，而不是DSRNA和DSDNA。结果已发表在EmboJ。 为了捕获与其底物复合的病毒RNase H的晶体结构，我们产生了全长的HIV逆转录酶和RNase H催化突变体。特殊的RNA/DNA底物的设计目的是针对HIV RT结晶试验的RNase活性位点。与NCI的Stuart Le Grice小组合作，我们在RNase H活性位点与RNA/DNA底物的共晶体启动了HIV RT变体和RNA/DNA混合体的广泛屏幕。 参考 诺特尼（M.人RNase H1的结构与RNA/DNA杂种复合：对HIV逆转录的见解。摩尔。 Cell，28，264-276。