Aids Related Nmr Research

艾滋病相关核磁共振研究

基本信息

批准号：
7968062
负责人：
Robert E London
金额：
$ 115.3万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
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/7968062
关键词：
Acquired Immunodeficiency Syndrome Active Sites Address Adopted Affect Affinity Agreement Area Base Excision Repairs Behavior Binding Biological Models Calmodulin Cells Characteristics Chemicals Complex Coupling DBL Oncoprotein DNA DNA Polymerase beta Data Data Reporting Databases Dependence Development Dimerization Drug Delivery Systems Environment Enzymes Evaluation Fingers Genetic Transcription HIV HIV-1 Human Hybrids Intervention Investigation Label Ligand Binding Ligands Literature Malignant Neoplasms Methionine Methods Modeling Molecular Molecular Conformation Monitor Mutation NMR Spectroscopy Nevirapine Nuclear Magnetic Resonance Nucleotides Oxygen Pathway interactions Pharmaceutical Preparations Polymerase Process Proteins RNA RNA-Directed DNA Polymerase Relative (related person)Relaxation Research Retroviridae Ribonuclease H Roentgen Rays Signal Transduction Site Site-Directed Mutagenesis Solutions Solvents Structure Study models Suggestion Sulfides Sulfur System Techniques Thumb structure Tumor Suppression Viral Genome base design dimer ds-DNA grasp inhibitor/antagonist interest methionine methyl ester methyl group methyl tert-butyl ether mutant non-nucleoside reverse transcriptase inhibitors nuclease nucleoside inhibitor

项目摘要

This project utilizes NMR spectroscopy to study the molecular components of HIV and model systems. The primary research areas are: 1) analysis of the structure, dynamics and ligand binding behavior of HIV reverse transcriptase and its RNase H domain, 2) related NMR methodological evaluations and development that facilitates the design and interpretation of the RT studies; 3) studies of model nuclease and polymerase systems, particularly DNA pol beta. Project 1. HIV Reverse Transcriptase (RT) is a primary target for drug intervention in the treatment of AIDS. We have performed the first NMR studies of methyl-13C methionine HIV-1 RT, aimed at better understanding the conformational and dynamic characteristics of RT, both in the presence and absence of the non-nucleoside RT inhibitor (NNRTI) nevirapine. The selection of methionine as a structural probe was based both on its favorable NMR characteristics, and on the presence of two important active site methionine residues in the p66 subunit: M184 and M230. Observation of the M184 resonance is subunit dependent; in the p66 subunit the solvent-exposed residue produces a readily observed signal with a characteristic resonance shift, while in the globular p51 subunit, the M184 resonance is shifted and broadened as M184 becomes buried in the protein interior. In contrast, although structural data indicates that the environment of M230 is also strongly subunit dependent, the M230 resonances from both subunits have very similar shift and relaxation characteristics. A comparison of chemical shift and intensity data with model-based predictions for the p66 subunit gives reasonable agreement for M184, while M230, located on the -hairpin "primer grip", is more mobile and solvent exposed than suggested by crystal structures of the apo enzyme which have a "closed" fingers-thumb conformation. This mobility of the primer grip is presumably important for binding of non-nucleoside RT inhibitors (NNRTIs), since the NNRTI binding pocket is not observed in the absence of the inhibitors, requiring instead that the binding pocket be dynamically accessible. In the presence of the nevirapine, the resonances of M184 and M230 in the p66 subunit are both significantly perturbed, while none of the methionine resonances in the p51 subunit is sensitive to this inhibitor. Site-directed mutagenesis indicates that both M16 and M357 produce two resonances in each subunit, and for both residues, the intensity ratio of the component peaks is strongly subunit dependent. Project 2. Dimerization of the p51 subunit of HIV reverse transcriptase. The dimerization of HIV reverse transcriptase has been of interest since it is significantly influenced by non-nucleoside RT inhibitors (NNRTI) and is therefore presumably related to their mechanism of action, and since the development of dimerization inhibitors has also become an active field for drug research. Our recent study utilized methyl-13Cmethionine labeling as well as small angle X-ray scattering (SAXS) to evaluate the dimerization of the p51 subunit, and to understand how this process relates to the conformational behavior of the p51 subunit. As demonstrated recently, the methionine resonances of methyl-13Cmethionine RT show significant subunit dependence, which is generally consistent with the large structural differences of the p51 and P66 subunits. The 1H-13C HSQC spectrum of methyl-13Cmethionine-labeled p51 is qualitatively similar to that expected for a mixture of the p66 and p51 subunits, indicating that a significant fraction of the p51 adopts a "p66-like" conformation. Additional NMR and SAXS studies indicate that under the conditions of our studies, p51 exists primarily as a homodimer that is a conformational heterodimer. The effects of the NNRTI nevirapine and Mg concentration on both the dimerization and the conformation also have been evaluated. Project 3. The approach of selective labeling of RT with methionine described above has proven useful for the investigation of this large and complex molecule, but also provides less information than a more general labeling approach would yield. We have been interested in optimizing the information available from such studies. During the past year, we have performed DFT calculations on model systems in order to evaluate the conformational dependence of 3JCSCC, 3JCSCH, and the isotropic shielding of the 13C methyl group. Results have been compared with experimental data reported in the literature, as well as data obtained on methyl-13Cmethionine and on the model compounds t-butyl-methyl sulfide and t-butyl methyl ether. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, 3JCSCC /3JCOCC is about .76. In addition it is demonstrated that the 3JCSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range for methionine CΗ3 in the TALOS data base supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of the methionine methyl 13C shift. Analysis of calmodulin data based on these calculations indicates that a few residues adopt non-standard gauche rotamers characterized by very large chi3 values. The utility of the methionine methyl 13C shift as a basis for estimating the gauche/trans ratio for chi3 has been evaluated, and supports our conclusion that Met16 of HIV reverse transcriptase is primarily in the trans conformation. Project 4. DNA polymerase beta has been considered as a model for the behavior of HIV reverse transcriptase. We have previously demonstrated that conformational activation of Pol beta can be readily monitored by the methionine resonances which show characteristic perturbations upon the binding of gapped DNA substrates as well as on the formation of an abortive ternary complex. During the past year, we have extended our studies of cancer associated Pol beta mutants in order to better understand the phenotypic consequences of these mutations.

该项目利用NMR光谱研究HIV和模型系统的分子成分。主要研究领域是：1）分析HIV逆转录酶及其RNase H结构域的结构，动力学和配体结合行为，2）相关的NMR方法论评估和发展，从而促进RT研究的设计和解释； 3）模型核酸酶和聚合酶系统，尤其是DNA POLβ的研究。项目1。HIV逆转录酶（RT）是药物干预艾滋病治疗的主要目标。在存在和不存在非核苷RT抑制剂（NNRTI）奈维罗嗪的情况下，我们进行了第一个NMR的NMR研究，旨在更好地理解RT的构象和动态特征。蛋氨酸作为结构探针的选择既基于其有利的NMR特征，又基于p66亚基中两个重要的活性位点蛋氨酸残基的存在：M184和M230。 M184共振的观察是亚基依赖性的。在p66亚基中，暴露于溶剂的残基会产生一个容易观察到的信号，具有特征性的共振转移，而在球状p51亚基中，随着M184被埋葬在蛋白质内部中，M184共振会发生变化和扩展。相反，尽管结构数据表明M230的环境也很强，但两个亚基的M230共振具有非常相似的变化和放松特征。化学位移和强度数据与基于模型的p66亚基的比较给出了M184的合理一致性，而位于-Hairpin“引物握把”上的M230比具有“封闭的”手指的thumber-thumb构象的Apo酶的晶体结构所建议的更可移动和溶剂。底漆握把的这种迁移率可能对非核苷RT抑制剂（NNRTIS）结合而言很重要，因为在没有抑制剂的情况下未观察到NNRTI结合袋，而是要求结合袋可以动态访问。在奈韦拉平的存在下，p66亚基中M184和M230的共振均显着扰动，而p51亚基中的蛋氨酸共振均对此抑制剂敏感。定点诱变表明M16和M357在每个亚基中都产生两个共振，对于两个残基，组件峰的强度比非常依赖于亚基。项目2。HIV逆转录酶的p51亚基的二聚化。 HIV逆转录酶的二聚化引起了人们的关注，因为它受到非核苷RT抑制剂（NNRTI）的显着影响，因此可能与其作用机理有关，并且由于二聚化抑制剂的发展也已成为药物研究的活性领域。我们最近的研究利用甲基-13cmethionine标记以及小角度X射线散射（SAX）来评估p51亚基的二聚化，并了解该过程如何与p51亚基的构象行为相关联。如最近所示，甲基-13cmethionine rt的蛋氨酸共振显示出显着的亚基依赖性，这通常与p51和p66亚基的较大结构差异一致。甲基-13cmethion-label的p51的1H-13C HSQC光谱在质量上与P66和p51亚基的混合物的预期相似，表明p51的大部分都采用了“ p66类似”的构象。其他NMR和SAXS研究表明，在我们的研究条件下，p51主要作为均二聚体，是一种构象异二聚体。还评估了NNRTI奈韦拉平和Mg浓度对二聚化和构象的影响。项目3。上述RT选择性标记的方法已证明对研究这个大型且复杂的分子的研究很有用，但与更通用的标记方法相比，提供的信息也更少。我们有兴趣优化此类研究中可用的信息。在过去的一年中，我们对模型系统进行了DFT计算，以评估3JCSCC，3JCSCH和13C甲基的各向同性屏蔽的构象依赖性。结果已与文献中报道的实验数据以及在甲基-13cmethionsine和模型化合物上获得的数据进行了比较。这些研究表明，相对于氧，耦合途径中硫原子的存在导致耦合常数明显较小，3JCSCC /3JCOCC约为.76。另外，证明3JCSCH耦合常数主要取决于亚倾斜的CSCH二面角，其次是CSCC二面角。将理论屏蔽计算与TALOS数据库中蛋氨酸Cη3的实验偏移范围的比较支持了以下结论：残留构象内依赖性的偏移扰动是甲基氨基甲基13C移位的主要决定因素。基于这些计算的钙调蛋白数据的分析表明，一些残基采用以非常大的CHI3值为特征的非标准的Gauche Rotamers。已经评估了蛋氨酸甲基13c移位作为估计CHI3的GAUCHE/反式比率的基础的效用，并支持我们的结论，即HIV逆转录酶的MET16主要在反式构象中。项目4。DNA聚合酶β已被视为HIV逆转录酶行为的模型。我们先前已经证明，POLβ的构象激活可以通过蛋氨酸的共振很容易监测，这些共振在间隙DNA底物的结合以及形成堕胎三元复合物的结合时表现出了特征性的扰动。在过去的一年中，我们扩展了对癌症相关的POL Beta突变体的研究，以便更好地了解这些突变的表型后果。