Specificity of acyl transfer from 2-mercaptobenzamide thioesters to the HIV-1 nucleocapsid protein
Specificity of acyl transfer from 2-mercaptobenzamide thioesters to the HIV-1 nucleocapsid protein
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DOI：
10.1021/ja071254o
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发表时间：
2007-09-12
影响因子：
15
通讯作者：
Appella, Ettore
中科院分区：
文献类型：
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作者：
Jenkins, Lisa M. Miller;Hara, Toshiaki;Appella, Ettore
HIV-1 核衣壳蛋白 (NCp7) 是一种高度保守的小蛋白，具有两个对该蛋白功能至关重要的锌结合结构域。目前正在评估与 NCp7 结合并使其失活的分子作为新的抗病毒药物。特别是，基于 2-巯基苯甲酰胺硫酯模板的衍生物已被证明可以通过从硫酯到半胱氨酸硫的酰基转移，从 NCp7 的 C 末端锌结合结构域 (ZD2) 特异性排出锌。在这项研究中，NCp7氨基酸序列的突变分析被用来研究ZD2和2-巯基苯甲酰胺硫酯化合物之间相互作用的特异性，使用紫外可见光谱和质谱法分别监测NCp7突变肽和酰化位点的金属喷射速率。我们能够扩展先前报道的这些硫酯化合物的作用机制，以包括在从硫酯到蛋白质中的半胱氨酸侧链的初级酰基转移之后发生的次级S到N分子内酰基转移。然后检查硫酯/ZD2 复合物的结构模型以确定最可能的结合方向。我们确定位置 x + 1（其中 x 是 CYs(36)）需要是芳香族残基才能实现反应性，而位置 x+9 上的氢键供体对于最佳反应性非常重要。为了正确折叠，位置 x + 2 需要碱性残基（赖氨酸或精氨酸），而涉及 S 到 N 酰基转移的反应需要赖氨酸残基。我们报告了 2-巯基苯甲酰胺硫酯化合物和 NCp7 之间高度特异性的相互作用，为改进和设计新的抗逆转录病毒疗法提供了结构基础，针对抗病毒突变的靶点。
The HIV-1 nucleocapsid protein (NCp7) is a small, highly conserved protein with two zinc-binding domains that are essential for the protein's function. Molecules that bind to and inactivate NCp7 are currently being evaluated as new antiviral drugs. In particular, derivatives based on a 2-mercaptobenzamide thioester template have been shown to specifically eject zinc from the C-terminal zinc-binding domain (ZD2) of NCp7 via acyl transfer from the thioester to a cysteine sulfur. In this study, mutational analysis of the NCp7 amino acid sequence has been used to investigate the specificity of the interaction between ZD2 and a 2-mercaptobenzamide thioester compound using UV-vis spectroscopy and mass spectrometry to monitor the rate of metal ejection from NCp7 mutant peptides and sites of acylation, respectively. We were able to extend the previously reported mechanism of action of these thioester compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine side chain in the protein. Structural models of the thioester/ZD2 complex were then examined to identify the most likely binding orientation. We determined that position x + 1 (where x is CYs(36)) needs to be an aromatic residue for reactivity and a hydrogen-bond donor in position x+9 is important for optimal reactivity. A basic residue (lysine or arginine) is required at position x + 2 for the correct fold, while a lysine residue is needed for reactivity involving S to N acyl transfer. We report highly specific interactions between 2-mercaptobenzamide thioester compounds and NCp7 that offer a structural basis for refining and designing new antiretroviral therapeutics, directed toward a target that is resistant to viral mutation.