Search for the Structural Basis of Biomacromolecular Function and Activity

寻找生物大分子功能和活性的结构基础

• 批准号: 7965286
• 负责人: Yun Xing m wang
• 金额: $ 131.57万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/7965286
• 关键词: Accounting; Acids; Amino Acids; Architecture; Biochemical; Biological; Biological Process; Biology; CCR; Cancer Biology; Clinic; Code; Collaborations; Complex; Computer Simulation; Data; Dimensions; Down-Regulation; Event; Family; Foxes; Gait; Gene Expression; Gene Expression Regulation; Genetic Transcription; Goals; HIV Protease; Indium; Journals; LIMS1 gene; Laboratories; Macromolecular Complexes; Mammalian Cell; Manuscripts; Maps; Measurement; Messenger RNA; Methodology; Methods; Molecular; NMR Spectroscopy; Noise; Nuclear; Nucleotides; Ornithine Decarboxylase; Pathway interactions; Planet Mars; Plant Viruses; Plants; Play; Polyamines; Process; Proteins; Publishing; Putrescine; RNA; RNA Binding; RNA-Protein Interaction; Relative (related person); Reporting; Research; Residual state; Resolution; Roentgen Rays; Role; Shapes; Signal Transduction; Simulate; Site; Solutions; Spermidine; Spermine; Staging; Structure; Surface; System; Testing; Transfer RNA; Translation Initiation; Translation Process; Translations; Tumor Angiogenesis; Turnip - dietary; Untranslated Regions; Up-Regulation; Vascular Endothelial Growth Factors; Vertebral column; Viral; Virus; angiogenesis; base; beta catenin; glutamyl-prolyl-tRNA synthetase; human VEGF protein; integrin-linked kinase; novel; professor; programs; prototype; receptor; restraint; structural biology; theories; three dimensional structure; tumor progression; turnip yellow mosaic virus; viral RNA

During the past year, we have been focusing to develop new methodologies that are needed for studying the structural biology of the 3' UTR RNA of the human VEGF mRNA. VEGF is a single most important protein that is critical to angiogenesis. These methods include GASR (J. Am. Chem. Soc., 2009, 131 (30), pp 10507-10515) and G2G (J. Mol. Biol., 2009, Aug. 8). GASR stands for "derive global architecture from SAXS and RDC" data, and G2G stands for "derive global structure of large RNAs in solution from global restraints". In addition, we have applied the G2G method to determine the three dimensional structure of the 3' UTR RNA fragment (102 nt) of the turnip crinkle virus (TCV). This 102-nt RNA plays an important role in enhancing translation initiation and is the prototype of 3' UTR RNAs that involved in regulation of gene expression. Expression of many proteins important to cancer biology, such as VEGF, is also controlled by 3' UTR RNAs. 1. Developing GASR method. Determining global structures of multi-subunit biomacromolecules in solution is a challenging problem. We report here a methodology that determines the global architecture of hetero- or homodimeric systems in solution using the residual dipolar couplings (RDCs) as the global orientation and the small-angle X-ray scattering (SAXS) as the global shape restraints; this methodology is implemented in an efficient program, known as global architecture derived from SAXS and RDC (GASR). We first applied the method to derive the global architecture of HIV protease, a globular homodimeric protein, to test the robustness of the method using simulated data with added noise. We then applied the method to determine the interfaces in the integrin-linked kinase (ILK)-PINCH complex using the experimental data. Without the benefit of SAXS data, the global architecture (and thus the interfaces of this complex) was underdetermined because of a lack of a sufficient number of detectable experimental distance restraints between the subunits of the complex. This method provides a new general approach for determining global structures of macromolecular complexes in solution. We have published the first manuscript on the method for an RNA:RNA complex (JACS, 2008 Mar 19;130(11):3292-3). An article that present a full theoretical and experimental description of the method has been published (J. Am. Chem. Soc., 2009, 131 (30), pp 10507-10515). 2. Developing G2G method. We have developed a novel top-down methodology that uses duplexes global orientations and overall molecular dimension restraints, which were extracted from experimental NMR and small angle X-ray scattering (SAXS) data respectively, to determine global architectures of large RNA molecules. The methodology is implemented in an efficient computational program package, G2G. We demonstrate the efficiency of the method by rapidly determining the global structure of a 71-nucleotide RNA using experimental data. The backbone RMSD of the ensemble of the calculated global structures relative to the X-ray crystal structure is 3.0 0.4 , and this RMSD is only 2.4 0.16 for the three duplexes that were orientation- and translation-restrained during the calculation. The global structure dramatically simplifies interpretation of multi-dimensional nuclear Overhauser spectra for a rapid high resolution structure determination. We anticipate that this top-down method will become the standard routine for determining structures of large RNAs in solution. An article that presents a full account of theory and experimental aspects of the method has been published in JMB (doi:10.1016/j.jmb.2009.08.001 ). 3. Structure determination of the TCV RNA using NMR and small angle X-ray scattering (SAXS). The 3′ UTRs of many plant viral mRNAs have been demonstrated to have tRNA-like activities and have been proposed to fold into a tRNA-like structure. Among these plant viral RNAs, the 3′ UTR of the turnip yellow mosaic virus (TYMV) was best characterized biochemically, and a tRNA-like computational model based on tRNA has been proposed. However, a three-dimensional structure has not been determined for any of these UTRs. The experimentally determined tRNA-like structures will provide a critical piece of evidence to support the notion that the 3′ UTRs of these plant viruses are directly involved in the translation process and may be considered a conceptual step toward understanding the general mechanisms of 3′ UTR involvement in the regulation of gene expression. The tRNA-like structure of a 3′ UTR would also provide a critical piece of evidence supporting the notion that the function of these 3′ UTRs is related to their ability to form tRNA-like structures. The involvement of the 3′ UTR in translation may not be limited to plant viruses. Two other examples found in mammalian cells are the 3′ UTR of mRNAs coding for the ornithine decarboxylase (ODC) and the vascular endothelial growth factor (VEGF). In mammalian cells, the polyamines putrescine, spermidine, and spermine are essential for proliferation and differentiation. The mammalian ODC catalyzes the first and rate-limiting steps in the polyamine biosynthetic pathway. VEGF plays an important role in tumor angiogenesis and cancer progression. Both of these essential proteins are regulated at multiple levels, including the mRNA level, via 3′ UTRs. It has been proposed by Dr. Bruce Shapiros laboratory, of the CCR, that both RNAs share a secondary structure similar to that of the turnip crinkle virus 3′ UTR fragment (tcvRNA), and therefore, possibly the tertiary structure. We have applied the G2G method to determine the global structure of the tcvRNA. A manuscript that describes the structure determination of the tcvRNA is currently under consideration of a journal. 3. The gene expression of VEGF is regulated at multiple stages, including but not limited to post-transcription and translational levels. The down-regulation of the VEGF expression has been mapped to specific interactions between RNA125 and one of proteins in the GAIT complex. An earlier study by Dr. Fox's laboratory showed that the down-regulation involves specific interactions between the linker domain of the glutamyl-prolyl tRNA synthetase (EPRS), which is one of components of the GAIT complex, and a 29-nt hairpin (H29) that is entirely composed of A or U nucleotides and is the part of the 125-nt RNA . The structural basis for this important interaction is unknown. The EPRS linker domain (ELD) consists of three dual-helical bundles and the interaction site for H29 has been mapped to the first two helical bundles, r1r2, by Dr. Fox's group using biochemical methods. r1r2 contains 114 amino acids and the three-dimensional structure of the first dual-helical bundle has been reported. The helical structure of r1r2 differs from known RNA-binding structural motifs and may represent a new RNA-binding motif. We have completed assigning the backbone signals of r1r2 and are in the process of mapping the RNA/protein interaction surface. We are currently investigating the structural basis of down- and up-regulation of the VEGF expression using combination of NMR and SAXS. We have established a collaboration with Professor Paul Fox's group of the Cleveland Clinic to corroborate our structural results with biological functions.

在过去的一年里，我们一直专注于开发新的方法，用于研究人类VEGF mRNA的3' UTR RNA的结构生物学。VEGF是一种最重要的蛋白质，对血管生成至关重要。这些方法包括GASR (J. Am.；化学。Soc。[j] .生物工程学报，2009,31 (30),pp 10507-10515 .]， 2009年8月8日)。GASR代表“从SAXS和RDC数据中推导出全局架构”，G2G代表“从全局约束中推导出解决方案中大rna的全局结构”。此外，我们还应用G2G方法测定了芜菁皱病毒（TCV） 3' UTR RNA片段（102 nt）的三维结构。这种102-nt RNA在促进翻译起始中起重要作用，是参与基因表达调控的3' UTR RNA的原型。许多对癌症生物学很重要的蛋白质的表达，如VEGF，也受到3' UTR rna的控制。1. 开发GASR方法。确定溶液中多亚基生物大分子的整体结构是一个具有挑战性的问题。我们在这里报告了一种方法，该方法确定了溶液中异二聚体或同二聚体系统的整体结构，使用残余偶极耦合（rdc）作为整体取向，小角x射线散射（SAXS）作为整体形状约束；这种方法是在一个高效的程序中实现的，称为从SAXS和RDC派生的全局架构（GASR）。我们首先将该方法应用于推导HIV蛋白酶（一种球状同型二聚体蛋白）的全局结构，并使用添加噪声的模拟数据测试该方法的鲁棒性。然后，我们利用实验数据应用该方法确定了整合素连接激酶(ILK)-PINCH复合物中的界面。没有SAXS数据的好处，由于复合物亚基之间缺乏足够数量的可检测实验距离限制，因此复合物的整体结构（以及该复合物的界面）尚未确定。该方法为测定溶液中大分子配合物的整体结构提供了一种新的通用方法。我们已经发表了第一篇关于RNA:RNA复合物方法的手稿（JACS， 2008年3月19日；130(11):3292-3）。已经发表了一篇文章，对该方法进行了完整的理论和实验描述。化学。Soc。中文信息学报，2009,131 (30),pp 10507-10515)。2. 开发G2G方法。我们开发了一种新颖的自顶向下的方法，该方法使用分别从实验NMR和小角度x射线散射（SAXS）数据中提取的双分子全局取向和总体分子尺寸限制来确定大RNA分子的全局结构。该方法是在一个高效的计算程序包G2G中实现的。我们通过使用实验数据快速确定71核苷酸RNA的整体结构来证明该方法的效率。计算得到的整体结构系综相对于x射线晶体结构的主链RMSD为3.0 0.4，而在计算过程中受取向和平移限制的3个双相系的主链RMSD仅为2.4 0.16。整体结构极大地简化了多维核奥弗豪泽光谱的解释，从而实现了快速的高分辨率结构确定。我们预计这种自上而下的方法将成为确定溶液中大rna结构的标准程序。在JMB上发表了一篇文章，全面介绍了该方法的理论和实验方面（doi:10.1016/j.j JMB .2009.08.001）。3. 利用核磁共振和小角x射线散射（SAXS）测定TCV RNA的结构。3, # 8242;许多植物病毒mrna的utr已被证明具有trna样活性，并被提出折叠成trna样结构。在这些植物病毒rna中，3&#8242；芜菁黄花叶病毒（turnip yellow mosaic virus， TYMV）的UTR在生物化学上得到了最好的表征，并提出了基于tRNA的类tRNA计算模型。然而，尚未确定任何这些utr的三维结构。实验确定的trna样结构将提供一个关键的证据来支持这一观点：这些植物病毒的utr直接参与转译过程，可能被认为是理解这些病毒的一般机制的一个概念性步骤。UTR参与基因表达的调控。3&#8242；UTR还将提供一个关键的证据来支持这样一种观点，即这些基因的功能utr与它们形成trna样结构的能力有关。3&#8242；翻译后的UTR可能不限于植物病毒。在哺乳动物细胞中发现的另外两个例子是3&amp；编码鸟氨酸脱羧酶（ODC）和血管内皮生长因子（VEGF）的mrna的UTR。在哺乳动物细胞中，多胺腐胺、亚精胺和精胺是增殖和分化所必需的。哺乳动物ODC催化多胺生物合成途径的第一步和限速步骤。VEGF在肿瘤血管生成和肿瘤进展中起重要作用。这两种必需蛋白质都在多个水平上受到调节，包括mRNA水平，通过3&amp；#8242；utr。CCR的Bruce Shapiros博士实验室提出，这两种rna的二级结构与芜菁皱缩病毒的二级结构相似。UTR片段（tcvRNA），因此，可能是三级结构。我们应用G2G方法来确定tcvRNA的全局结构。一份描述tcvRNA结构测定的手稿目前正在某期刊的考虑中。3. VEGF的基因表达受到多个阶段的调控，包括但不限于转录后和翻译水平。VEGF表达的下调已被定位为RNA125与步态复合体中的一种蛋白质之间的特定相互作用。Fox博士实验室的一项早期研究表明，下调涉及谷氨酰脯氨酸tRNA合成酶（EPRS）的连接域（EPRS是步态复合物的组成部分之一）与29 nt发夹（H29）之间的特定相互作用，H29完全由a或U核苷酸组成，是125 nt RNA的一部分。这种重要相互作用的结构基础尚不清楚。EPRS连接结构域（ELD）由三个双螺旋束组成，Fox博士的团队使用生化方法将H29的相互作用位点映射到前两个螺旋束r1r2上。R1r2含有114个氨基酸，第一个双螺旋束的三维结构已被报道。r1r2的螺旋结构不同于已知的rna结合结构基序，可能是一种新的rna结合结构基序。我们已经完成了r1r2的骨干信号的分配，并正在绘制RNA/蛋白相互作用表面。我们目前正在利用NMR和SAXS联合研究VEGF表达下调和上调的结构基础。我们已经与克利夫兰诊所的保罗·福克斯教授小组建立了合作关系，以证实我们的结构结果与生物学功能。