Ascone I, Lenouvel F, Sequeval D, Dexpert H, Felenbok B
Laboratoire d'Utilisation du Rayonnement Electromagnétique, Faculté d'Orsay, France.
Biochim Biophys Acta. 1997 Dec 5;1343(2):211-20. doi: 10.1016/s0167-4838(97)00112-x.
AlcR is the transcriptional activator of the ethanol utilization pathway in Aspergillus nidulans. The zinc DNA-binding domain contains ligands of zinc, six cysteines (Zn2Cys6) or five cysteines and one histidine (Zn2Cys5His). The utilisation of complementary approaches such as X-ray absorption spectroscopy, mutational analysis, zinc content evaluation, determination of specific binding connecting structural and biological data, have allowed to determine zinc environment and to analyse the involvement of amino acids. The determination by EXAFS of zinc ligands (four sulphur atoms), the Zn content in the protein (2:1), the evaluation of the distance between two zinc atoms (3.16 +/- 0.02 angstroms), together with the total loss of specific DNA-binding activity when one cysteine ligand is mutated, are in favour of a zinc cluster model in which six cysteine sulphurs ligate two zinc atoms. XANES spectra of wild type and H10A AlcR protein are virtually identical indicating that Histidine 10 does not have a direct contribution in zinc ligation but electrophoretic mobility shift assays show that His10 is involved in DNA-binding. In contrast, proline 25 does not seem to play any direct role in the DNA-binding activity but XANES spectra of Pro25A AlcR protein are slightly modified comparing to the wild type protein spectra. This suggests a role of the proline in the stabilisation of the Zn cluster structure. AlcR DNA-binding domain belongs to the zinc binuclear class family (Zn2Cys6) with unique characteristics resulting from its primary and secondary structures and its binding specificity toward direct and inverted repeat target.
AlcR是构巢曲霉乙醇利用途径的转录激活因子。锌DNA结合结构域包含锌的配体，六个半胱氨酸（Zn2Cys6）或五个半胱氨酸和一个组氨酸（Zn2Cys5His）。采用诸如X射线吸收光谱、突变分析、锌含量评估、确定连接结构和生物学数据的特异性结合等互补方法，已能够确定锌环境并分析氨基酸的作用。通过扩展X射线吸收精细结构（EXAFS）确定锌配体（四个硫原子）、蛋白质中的锌含量（2:1）、两个锌原子之间的距离评估（3.16±0.02埃），以及当一个半胱氨酸配体发生突变时特异性DNA结合活性的完全丧失，都支持锌簇模型，即六个半胱氨酸硫原子连接两个锌原子。野生型和H10A AlcR蛋白的X射线吸收近边结构（XANES）光谱几乎相同，表明组氨酸10在锌配位中没有直接作用，但电泳迁移率变动分析表明His10参与DNA结合。相比之下，脯氨酸25似乎在DNA结合活性中不发挥任何直接作用，但与野生型蛋白光谱相比，Pro25A AlcR蛋白的XANES光谱略有改变。这表明脯氨酸在锌簇结构的稳定中起作用。AlcR DNA结合结构域属于锌双核类家族（Zn2Cys6），因其一级和二级结构以及对直接和反向重复靶标的结合特异性而具有独特特征。
