Miller Jenkins Lisa M, Hara Toshiaki, Durell Stewart R, Hayashi Ryo, Inman John K, Piquemal Jean-Philip, Gresh Nohad, Appella Ettore
Laboratory of Cell Biology, NCI, and Laboratory of Immunology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Am Chem Soc. 2007 Sep 12;129(36):11067-78. doi: 10.1021/ja071254o. Epub 2007 Aug 18.
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 Cys36) 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.
HIV-1核衣壳蛋白（NCp7）是一种小的、高度保守的蛋白质，具有两个锌结合结构域，这两个结构域对该蛋白质的功能至关重要。目前正在评估与NCp7结合并使其失活的分子作为新型抗病毒药物。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通过硫酯向半胱氨酸硫的酰基转移，从NCp7的C端锌结合结构域（ZD2）特异性地排出锌。在本研究中，利用NCp7氨基酸序列的突变分析，通过紫外可见光谱和质谱分别监测从NCp7突变肽中排出金属的速率和酰化位点，来研究ZD2与2-巯基苯甲酰胺硫酯化合物之间相互作用的特异性。我们能够将这些硫酯化合物先前报道的作用机制扩展到包括在硫酯向蛋白质中半胱氨酸侧链进行初次酰基转移后发生的二级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为Cys36）需要是一个芳香族残基才能具有反应活性，而位置x + 9处的氢键供体对于最佳反应活性很重要。为了形成正确的折叠，位置x + 2需要一个碱性残基（赖氨酸或精氨酸），而涉及S到N酰基转移的反应活性则需要一个赖氨酸残基。我们报道了2-巯基苯甲酰胺硫酯化合物与NCp7之间高度特异性的相互作用，为改进和设计针对对病毒突变具有抗性的靶点的新型抗逆转录病毒疗法提供了结构基础。
