study of the HIV integrase catalytic core domain and inhibitor binding

HIV整合酶催化核心结构域和抑制剂结合的研究

• 批准号: 8148947
• 负责人: Ad Bax
• 金额: $ 29.13万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY 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/8148947
• 关键词: Binding; Catalytic Domain; HIV Integrase; inhibitor/antagonist

Through extensive mutagenesis trial-and-error efforts, a stable, homodimeric construct of the catalytic core domain of the HIV1 integrase enzyme has been generated. Although under conditions that closely resemble the physiological environment the enzyme rapidly samples different conformational states, leading to extensive line broadening and disappearance of NMR signals, we have found conditions that include high Mg2+ concentrations (40 mM) where the equilibrium is shifted to what appear to be one major and one minor state, that differ from one another in the structure of the C-terminal helix. The NMR chemical shifts are found to corroborate structures observed in crystals, and confirm prior studies suggesting that the α4 helix extends toward the active site. The strong improvement in spectral quality with Mg2+ concentration suggests a structural transition not only near the active site residues but also throughout the entire molecule as IN binds Mg2+. In particular, the stability of the core domain is linked to the conformation of its C-terminal helix, which has implications for relative domain orientation in the full length enzyme. 15N relaxation experiments further show that, while conformationally flexible, the catalytic loop of IN is not fully disordered in the absence of DNA. Indeed, automated chemical shift-based modeling of the active site loop reveals several stable clusters which show striking similarity to a recent crystal structure of prototype foamy virus (PFV) IN bound to DNA.

通过广泛的诱变试验努力，已经产生了HIV1整合酶催化核心结构酶的稳定，同型二聚体结构。 尽管在与生理环境紧密相似的条件下，酶迅速采样了不同的构象状态，从而导致NMR信号的广泛范围扩大和消失，我们发现了包括高MG2+浓度（40 mm）的条件，在这些条件下，平衡均转移到了一个主要和一个次要状态，与C-Cy-Ornix型架子的结构不同。 发现NMR化学位移可证实在晶体中观察到的结构，并证实了先前的研究，表明α4螺旋延伸到活性位点。 Mg2+浓度的光谱质量的强烈改善表明，不仅在活性位点残基附近，而且在整个分子中也像结合Mg2+一样。 特别是，核心结构域的稳定性与其C末端螺旋的构象相关，这对全长酶中的相对域取向具有影响。 15N松弛实验进一步表明，虽然在构象上柔性，但在没有DNA的情况下，IN的催化环并未完全无序。 实际上，主动位点环的自动化学基于化学移位的建模揭示了几个稳定的簇，这些簇与与DNA结合的原型泡沫病毒（PFV）的最新晶体结构相似。