MULTI-DIMENSIONAL NMR OF HIV-1 ENVELOPE GLYCOPROTEINS

HIV-1 包膜糖蛋白的多维核磁共振

• 批准号: 7681775
• 负责人: JACOB ANGLISTER
• 金额: $ 19.71万
• 依托单位: WEIZMANN INSTITUTE OF SCIENCE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7681775
• 关键词: Anti-HIV Agents; Antigens; Binding; Binding Sites; C-terminal; CCR5 gene; CXCR4 gene; Cell membrane; Cells; Complex; Educational process of instructing; Future; Fuzeon; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; Immune system; Individual; Label; Learning; Light; Mammalian Cell; Maps; Mediating; Membrane Glycoproteins; Molecular Conformation; N-terminal; Peptide T; Peptides; Pharmaceutical Preparations; Play; Proteins; Research; Research Personnel; Role; Solutions; Stream; Structure; Surface; T-20; T-Lymphocyte; Techniques; Viral; Virus; base; chemokine receptor; design; flexibility; inhibitor/antagonist; macrophage; novel; programs; receptor; receptor binding

DESCRIPTION (provided by applicant): The long term goals of our research are to learn about the mechanism for HIV-1 target recognition, to understand the selectivity of the virus to different cells of the immune system and to study mechanisms of viral entry inhibition. The binding of human immunodeficiency-virus type-1 (HIV-1) to its target cells is mediated primarily by the envelope glycoprotein (gp120) of the virus. Initially gp120 binds to CD4, a molecule found on the surface of both T-cells and macrophages. This binding triggers a conformational change in gp120 that forms another binding site to either CCR5 or CXCR4 chemokine receptors, which serve as co-receptors for HIV-1 binding. After binding to the co-receptor, gp120 undergoes another conformational change that exposes the gp41 trans-membrane glycoprotein of the virus which mediates HIV- 1 fusion with its target cell membrane. The peptide T-20 (Fuzeon), used as a viral entry inhibitor for the treatment of HIV-1 infected individuals, corresponds in its sequence to the C-terminal half of the gp41 region HR2 and a segment down stream of HR2. The structure of the co-receptor binding site on gp120 is still elusive and the mechanism for co-receptor selectivity is still unknown. Similarly, for T-20, the mechanism by which it inhibits fusion is not understood. Particularly enigmatic is the role played by its nine C-terminal residues, without which this anti-HIV drug loses its potency in entry-inhibition. The specific aims of the current proposal are as follows: a) Learn how the flexibility of gp120 enables the formation of the binding site for CD4 and CCR5, b) Learn how HIV-1 co-receptor selectivity is determined, and c) Investigate the interactions of T-20 with possible targets on gp41 and gp120 and reveal the role of the C-terminal segment T-20 in viral inhibition. We will use a novel multi-dimensional NMR technique designed to solve the structure of large proteins without requiring deuteration. The solution structure of HIV-1 gp120 in the unliganded form, in complex with a CD4-mimic peptide (miCD4) and in ternary complex with miCD4 and CCR5 extra-cellular fragments will be determined. In addition, we will determine the structure of the anti-HIV-1 drug T-20 in complex with gp41 and gp120 targets. When solved, the solution structure of HIV-1 gp120 will be probably the first structure of a protein expressed and labeled in mammalian cells and one of the largest structures solved by NMR. Considering the flexibility of gp120 that is directly related to its function, structure determination in solution is of utmost importance. The structure of gp120 in its three different conformations will enable us to learn about the mechanism for CD4 and co-receptor recognition and for designing anti-HIV- 1 immunogens, antagonists and entry inhibitors that target the gp120 and CCR5 binding sites. Studies of T- 20 complexes with gp41 and gp120 segments will reveal the role of the C-terminal segment of T-20 in viral inhibition and contribute to the design more potent entry inhibitors.

描述（由申请人提供）：我们研究的长期目标是了解HIV-1靶标识别的机制，了解病毒对免疫系统不同细胞的选择性，并研究病毒进入抑制的机制。人免疫缺陷病毒1型（HIV-1）与其靶细胞的结合主要由病毒的包膜糖蛋白（gp 120）介导。最初，gp 120与CD 4结合，CD 4是一种在T细胞和巨噬细胞表面发现的分子。这种结合引发了gp 120的构象变化，形成了与CCR 5或CXCR 4趋化因子受体的另一个结合位点，CCR 5或CXCR 4趋化因子受体是HIV-1结合的辅助受体。在与共受体结合后，gp 120经历另一种构象变化，暴露了介导HIV- 1与其靶细胞膜融合的病毒的gp 41跨膜糖蛋白。肽T-20（Fuzeon）用作治疗HIV-1感染个体的病毒进入抑制剂，其序列对应于gp 41区HR 2的C-末端一半和HR 2下游的片段。gp 120上的共受体结合位点的结构仍然是难以捉摸的，共受体选择性的机制仍然是未知的。类似地，对于T-20，其抑制融合的机制尚不清楚。特别神秘的是它的9个C-末端残基所起的作用，没有这些残基，这种抗HIV药物就会失去其进入抑制的效力。目前的建议的具体目标如下：a）了解gp 120的灵活性如何使CD 4和CCR 5的结合位点的形成，B）了解如何确定HIV-1共受体的选择性，和c）研究T-20与gp 41和gp 120上的可能靶点的相互作用，并揭示C-末端片段T-20在病毒抑制中的作用。我们将使用一种新的多维NMR技术，旨在解决大蛋白质的结构，而不需要氘代。将测定未配体形式、与CD 4模拟肽（miCD 4）复合以及与miCD 4和CCR 5胞外片段三元复合的HIV-1 gp 120的溶液结构。此外，我们还将确定抗HIV-1药物T-20与gp 41和gp 120靶标复合物的结构。一旦解决，HIV-1 gp 120的溶液结构可能是哺乳动物细胞中表达和标记的蛋白质的第一个结构，也是NMR解决的最大结构之一。考虑到与其功能直接相关的gp 120的灵活性，溶液中的结构确定至关重要。gp 120的三种不同构象的结构将使我们能够了解CD 4和辅助受体识别的机制，并设计针对gp 120和CCR 5结合位点的抗HIV- 1免疫原、拮抗剂和进入抑制剂。研究T- 20与gp 41和gp 120片段的复合物将揭示T-20的C端片段在病毒抑制中的作用，并有助于设计更有效的进入抑制剂。