Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry
Identification of an inhibitor-binding site to HIV-1 integrase with affinity acetylation and mass spectrometry
复制标题
DOI：
10.1073/pnas.0400873101
复制
发表时间：
2004-05-04
影响因子：
11.1
通讯作者：
Kvaratskhelia, M
中科院分区：
文献类型：
--
作者：
Shkriabai, N;Patil, SS;Kvaratskhelia, M
我们报告了一种结合亲和乙酰化和MS分析的方法，用于准确地将抑制物结合位点映射到目标蛋白。为此，我们使用了一种已知的含有芳基二-O-乙酰基的HIV-1整合酶抑制剂(乙酰化抑制剂)。此外，我们设计了一种对照化合物(乙酰化对照)，该化合物也含有芳基二O-乙酰基，但不抑制HIV-1整合酶。用包含所有20种天然氨基酸的模型肽库检测这些化合物的反应活性，发现芳基二氧-乙酰基化合物有效地乙酰化半胱氨酸、赖氨酸和酪氨酸残基。乙酰化抑制剂和乙酰化对照对这些小肽表现出类似的化学反应活性。然而，这两个化合物在与HIV-1整合酶的相互作用方面存在显著差异。特别是，乙酰化抑制剂在其抑制浓度(3um)下特异性地使K173乙酰化，而这个位置仍然不被乙酰化对照识别。我们的数据使我们能够为整合酶：乙酰化-抑制物复合体建立一个详细的模型，这表明抑制物选择性地结合在蛋白质的结构关键区域。本文所报道的方法对于涉及各种配基-蛋白质相互作用的系统具有普遍的应用。
We report a methodology that combines affinity acetylation with MS analysis for accurate mapping of an inhibitor-binding site to a target protein. For this purpose, we used a known HIV-1 integrase inhibitor containing aryl di-O-acetyl groups (Acetylated-inhibitor). In addition, we designed a control compound (Acetylated-Control) that also contained an aryl di-O-acetyl group but did not inhibit HIV-1 integrase. Examination of the reactivity of these compounds with a model peptide library, which collectively contained all 20 natural amino acids, revealed that aryl di-O-acetyl compounds effectively acetylate Cys, Lys, and Tyr residues. Acetylated-inhibitor and Acetylated-Control exhibited comparable chemical reactivity with respect to these small peptides. However, these two compounds differed markedly in their interactions with HIV-1 integrase. In particular, Acetylated-Inhibitor specifically acetylated K173 at its inhibitory concentration (3 muM) whereas this site remained unrecognized by Acetylated-Control. Our data enabled creation of a detailed model for the integrase:Acetylated-inhibitor complex, which indicated that the inhibitor selectively binds at an architecturally critical region of the protein. The methodology reported herein has a generic application for systems involving a variety of ligand-protein interactions.