Production Of HIV And HIV Related Proteins For Structural Studies

用于结构研究的 HIV 和 HIV 相关蛋白的生产

• 批准号: 8939414
• 负责人: PAUL T WINGFIELD
• 金额: $ 54.05万
• 依托单位: NATIONAL INSTITUTE OF ARTHRITIS AND MUSCULOSKELETAL AND SKIN 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/8939414
• 关键词: 3-Dimensional; Amino Acids; Anti-HIV Agents; Antibodies; Antibody Binding Sites; Bacteria; Binding; C-terminal; Cell Nucleus; Complex; Crystallization; Drug Design; Drug Targeting; Drug resistance; Epitopes; Exhibits; Filament; HIV; HIV Protease; HIV-1; Hot Spot; Immunoglobulin Fragments; Immunologic Deficiency Syndromes; Label; Life Cycle Stages; Light; Messenger RNA; Methods; Molecular Conformation; Molecular Structure; Mutation; Nuclear Magnetic Resonance; Organized by Structure Protein; Peptide Hydrolases; Peptides; Pharmaceutical Preparations; Production; Proteins; Recombinant DNA; Roentgen Rays; Role; Structural Protein; Structure; Testing; Time; Transcript; Viral; Virus; Work; X ray diffraction analysis; X-Ray Diffraction; base; genetic regulatory protein; novel; protein protein interaction; protein structure; rev Protein; stable isotope

Rev is a key regulatory protein of HIV-1. Its function is to bind to viral transcripts and effect export from the nucleus of unspliced mRNA, thereby allowing the production of structural proteins. In our previous work, the structure of Rev was solved for first time by complexing it with an antibody fragment (Fab). The anti-Rev Fab stabilized Rev allowing the formation of protein crystals suitable for X-ray structural analysis. The anti-Rev Fab was shown to have anti-HIV activity, presumably by binding tightly to Rev and blocking its functional interactions. Regions of the Fab antibody in contact with Rev are known collectively as the paratope (regions of Rev in contact with the Fab are known as the epitope). The paratope consists of six complementary determining region (CDR) loop-like regions, three each from the antibody heavy and light chains. The CDRs are relatively short consisting of 6 to 14 amino acid residues. Analyses of the epitope-paratope interface from the X-ray structure allowed for the prediction of key contacts (hot spots which bind most tightly) among the CDR sequences which stabilize the Rev: Fab complex. Peptides were synthesized which corresponded to the CDRs: the peptides were cyclized N- to C-terminal to give them a structure approximating to their loop-like conformation in the intact antibody. Of the CDRs peptides tested, one from the light chain (LCDR3) exhibited tight binding to Rev. Analogous to the antibody; the peptide was capable of depolymerizing a highly associated form of Rev (filaments) by specifically disrupting Rev protein-protein interactions. Mutations of residues in Fab, corresponding to the predicted hot spots in LCDR3, dramatically reduced its binding to Rev, confirming their key role in stabilizing the interaction. The LCDR3 peptide and other peptides selected for their binding to Rev are being assessed for their anti-HIV activity. HIV protease, a homodimeric protein, is essential in the viral life cycle and a major anti-HIV drug target. We have expressed and purified a number of wild type and drug resistant forms of the protease which have been used in structural studies. Novel drugs which bind to the protease have been studied by co-crystallization and by examining the crystal structures used to rationalize and optimize drug binding. Structural details of interactions between drug moieties and protein have led to the synthesis of new compound with higher anti-HIV potency.

Rev 是 HIV-1 的关键调节蛋白。其功能是与病毒转录物结合并影响未剪接的 mRNA 从细胞核输出，从而产生结构蛋白。在我们之前的工作中，通过将Rev与抗体片段（Fab）复合，首次解析了Rev的结构。抗 Rev Fab 稳定了 Rev，从而形成适合 X 射线结构分析的蛋白质晶体。抗 Rev Fab 被证明具有抗 HIV 活性，可能是通过与 Rev 紧密结合并阻断其功能相互作用来实现的。 Fab 抗体与 Rev 接触的区域统称为互补位（Rev 与 Fab 接触的区域称为表位）。互补位由六个互补决定区 (CDR) 环样区域组成，其中抗体重链和轻链各三个。 CDR 相对较短，由 6 至 14 个氨基酸残基组成。通过 X 射线结构对表位-互补位界面进行分析，可以预测稳定 Rev: Fab 复合物的 CDR 序列之间的关键接触点（结合最紧密的热点）。合成了与 CDR 相对应的肽：将肽从 N 端环化到 C 端，使其结构接近于完整抗体中的环样构象。在测试的 CDR 肽中，来自轻链 (LCDR3) 的一种表现出与抗体类似的 Rev 的紧密结合；该肽能够通过特异性破坏 Rev 蛋白-蛋白相互作用来解聚高度相关形式的 Rev（丝）。 Fab 中残基的突变（对应于 LCDR3 中预测的热点）显着降低了其与 Rev 的结合，证实了它们在稳定相互作用中的关键作用。 LCDR3 肽和其他因与 Rev 结合而选择的肽的抗 HIV 活性正在接受评估。 HIV 蛋白酶是一种同型二聚体蛋白，在病毒生命周期中至关重要，也是主要的抗 HIV 药物靶标。我们表达并纯化了许多野生型和耐药形式的蛋白酶，这些蛋白酶已用于结构研究。通过共结晶和检查用于合理化和优化药物结合的晶体结构，研究了与蛋白酶结合的新药物。药物部分和蛋白质之间相互作用的结构细节导致了具有更高抗 HIV 效力的新化合物的合成。