Timmerman J, Vuidepot A L, Bontems F, Lallemand J Y, Gervais M, Shechter E, Guiard B
Groupe de RMN-Département de Chimie, Synthèse Organique, Palaiseau, France.
J Mol Biol. 1996 Jun 21;259(4):792-804. doi: 10.1006/jmbi.1996.0358.
CYP1(HAP1) is a transcriptional activator involved in the aerobic metabolism of the yeast Saccharomyces cerevisiae. The amino acid sequence of its DNA-binding domain suggests that it belongs to the "zinc cluster" class. This region is indeed characterized by a pattern known to form a bimetal thiolate cluster where two zinc ions are coordinated by six cysteine residues. Structures of two such domains, those from GAL4 and PPR1, have been solved as complexes with DNA. These domains consist of the zinc cluster connected to a dimerization helix by a linker peptide. They recognize, as a dimer, an inverted repeat of a CGG motif that is separated by a specific number of bases. Interestingly, the specificity of that interaction seems not to be due to the interaction between the cluster region and the DNA but rather to a fine tune between the structure of the linker peptide and the number of base-pairs separating the two CGGs. However, the CYP1 target sites fail to display such a consensus sequence. One of the two CGG sites is poorly conserved and some experiments suggest a direct rather than an inverted repeat. Using 1H, 15N and 113Cd NMR spectroscopy, we have undertaken the analysis of the structural properties of the CYP1(56-126) fragment that consists of the zinc-cluster region, the linker peptide and a part of the dimerization helix. We have demonstrated that the six cysteine residues of the peptide chelate two cadmium ions as in GAL4 and PPR1. Fifteen structures of the zinc-cluster region (residues 60 to 100) were calculated, the linker peptide and the dimerization helix being unstructured under the conditions of our study. This region possesses the same overall fold as in GAL4 and PPR1, and most of the side-chains involved in the interaction with DNA are structurally conserved. This suggests that the CYP1 zinc-cluster region recognizes a CGG triplet in the same way as GAL4 and PPR1. In this case, the particular properties of CYP1 seem to be due to the structure of the linker peptide and/or of the dimerization helix.
CYP1（HAP1）是一种参与酿酒酵母有氧代谢的转录激活因子。其DNA结合结构域的氨基酸序列表明它属于“锌簇”类。该区域确实具有已知形成双金属硫醇盐簇的模式特征，其中两个锌离子由六个半胱氨酸残基配位。两个这样的结构域，即来自GAL4和PPR1的结构域，已被解析为与DNA的复合物结构。这些结构域由通过连接肽连接到二聚化螺旋的锌簇组成。它们作为二聚体识别被特定数量碱基隔开的CGG基序的反向重复序列。有趣的是，这种相互作用的特异性似乎不是由于簇区域与DNA之间的相互作用，而是由于连接肽的结构与分隔两个CGG的碱基对数量之间的精细调节。然而，CYP1靶位点未能显示出这样的共有序列。两个CGG位点中的一个保守性较差，一些实验表明是直接重复而非反向重复。利用1H、15N和113Cd核磁共振光谱，我们对CYP1（56 - 126）片段的结构特性进行了分析，该片段由锌簇区域、连接肽和部分二聚化螺旋组成。我们已经证明，该肽的六个半胱氨酸残基像在GAL4和PPR1中一样螯合两个镉离子。计算了锌簇区域（60至100位残基）的15种结构，在我们的研究条件下连接肽和二聚化螺旋是无结构的。该区域具有与GAL4和PPR1相同的整体折叠，并且与DNA相互作用中涉及的大多数侧链在结构上是保守的。这表明CYP1锌簇区域以与GAL4和PPR1相同的方式识别CGG三联体。在这种情况下，CYP1的特殊性质似乎归因于连接肽和/或二聚化螺旋的结构。
