Structure of Oligomeric HIV Rev-RRE Complexes

HIV Rev-RRE 寡聚复合物的结构

• 批准号: 7551271
• 负责人: Mirko Hennig
• 金额: $ 10.43万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 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/7551271
• 关键词: Affinity; Apis; Binding; Binding Sites; Biochemical; Biological Assay; Buffers; Calorimetry; Chemicals; Complex; Coupling; Data; Development; Diffusion; Fluorescence; Gel; HIV; HIV-1; Investigation; Label; Lead; Length; Ligands; Liquid substance; Maps; Measurement; Mediating; Messenger RNA; Methodology; Methods; Modems; Nature; Nuclear Export; Nuclear Magnetic Resonance; Numbers; Occupations; Physiologic pulse; Process; Proteins; Pulse taking; RNA; RNA-Protein Interaction; Rate; Relaxation; Resolution; Screening procedure; Solutions; Structure; Titrations; Variant; Viral; analog; analytical ultracentrifugation; base; dimer; fleephilone; fluoropyrimidine; follow-up; harziphilone; improved; inhibitor/antagonist; insight; light scattering; monomer; mutant; particle; preference; research study; small molecule; small molecule libraries; stem; stoichiometry; three dimensional structure

The nuclear export of HIV-1 viral mRNA is mediated by the activity of the regulator Rev. A feature of central importance of Rev function is the ability to self-associate. After Rev binds as a monomer to its primary high affinity RRE-binding site, a combination of cooperative protein-protein and protein-RNA interactions mediate the occupation of secondary binding sites resulting in oligomeric Rev-RRE complexes. Only oligomeric Rev-RRE complexes are nuclear export-competent. This proposal outlines structural studies using liquid state NMR methodology in combination with biochemical approaches that will reveal the detailed nature of the assembly of multiple Rev copies onto the RRE. 1) Characterization of Rev and Rev mutants self-association in the absence and presence of RRE. Rev fibrillizes in solution at concentrations required for liquid state NMR measurements. A screening of buffers facilitating structural analysis of Rev in solution will be performed. We want to identify oligomerization deficiant Rev mutants suitable for high-resolution NMR. We will verify the initial high affinity interaction with RRE stem-loop IIB and will determine stoichiometry of complexes formed by PAGE and ITC assays. 2) Structural Investigation of oligomeric Rev/RRE complexes in solution based on NMR methodology. We plan to solve the structure of Rev dimers providing insight into Rev's strong tendency to self-associate. Structural investigations of different target RRE constructs derived from stem II will be carried out. We will attempt to determine the structure of Rev mutants (monomeric/dimeric) bound to RRE stem-loop IIB and larger RRE variants (stem-loop II). 3) Characterization of structural features of Rev-RRE-inhibitor interactions focussing on ligand structures. We will attempt to determine the structure of the small molecule inhibitors, bound to either Rev and/or RRE using modem NMR methodology. We want to use 5-Fluoropyrimidine substituted RRE RNAs in directed screens utilizing 19F NMR. Based on the three-dimensional structure, we attempt to provide a lead structure for the rational development and synthesis of inhibitors with improved potency.

HIV-1 病毒 mRNA 的核输出是由调节因子 Rev 的活性介导的。中枢的一个特征 Rev 功能的重要性在于自我关联的能力。 Rev 作为单体与其初级高位结合后 亲和性 RRE 结合位点，蛋白质-蛋白质和蛋白质-RNA 相互作用的结合 介导二次结合位点的占据，从而形成寡聚 Rev-RRE 复合物。仅有的 Rev-RRE 寡聚复合物具有核输出能力。该提案概述了使用 液态核磁共振方法与生化方法相结合，将揭示详细的 将多个 Rev 副本组装到 RRE 上的性质。 1) 在 RRE 不存在和存在的情况下 Rev 和 Rev 突变体自关联的表征。牧师 在液态 NMR 测量所需的浓度下，在溶液中发生原纤维化。缓冲液的筛选 将进行溶液中 Rev 的结构分析。我们想要识别低聚 缺陷 Rev 突变体适合高分辨率 NMR。我们将验证初始的高亲和力交互 与 RRE 茎环 IIB 结合，将确定通过 PAGE 和 ITC 测定形成的复合物的化学计量。 2) 基于 NMR 方法对溶液中低聚 Rev/RRE 复合物进行结构研究。我们 计划解决 Rev 二聚体的结构，从而深入了解 Rev 的强烈自缔合倾向。 将对源自干 II 的不同目标 RRE 构建体进行结构研究。我们将 尝试确定与 RRE 茎环 IIB 结合的 Rev 突变体（单体/二聚体）的结构和 更大的 RRE 变体（茎环 II）。 3) Rev-RRE-抑制剂相互作用的结构特征表征，重点是配体结构。 我们将尝试确定与 Rev 和/或 RRE 结合的小分子抑制剂的结构 使用现代核磁共振方法。我们希望使用 5-氟嘧啶取代的 RRE RNA 来定向 利用 19F NMR 进行筛选。基于三维结构，我们尝试提供一种先导结构 合理开发和合成具有改进效力的抑制剂。