Metal and RNA binding properties of the hdm2 RING finger domain
Metal and RNA binding properties of the hdm2 RING finger domain
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DOI：
10.1021/bi980596r
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发表时间：
1998-12-01
期刊：
影响因子：
2.9
通讯作者：
McLendon, GL
中科院分区：
文献类型：
--
作者：
Lai, ZH;Freedman, DA;McLendon, GL
hdm2 癌蛋白含有一个结合 RNA 的 C 端结构域，并被认为可以在一个不寻常的环指结构域中结合锌 (II)，其中 Thr 455 被假定为配体。我们已经报道了测试这个富含半胱氨酸的 C 端基序是否确实是环指结构域的实验。我们还测试了 hdm2 C 端肽对金属结合的亲和力、与 C 端肽折叠的金属连接以及肽对 RNA 的亲和力。截短突变体证明氨基酸 425-491 对于 RNA 结合是必要且充分的。然而，二价金属离子似乎不影响特异性RNA识别。金属结合研究表明，hdm2 确实以类似于 BRCA1 环指肽的相互缠绕的基序与两个锌分子结合。然而，整体三级结构不存在相似性，与其他RING指也不存在直接序列同源性。荧光能量转移研究给出了钴 (II) 与位点 1 结合的解离常数为 (0.22 +/- 0.03) μM，而根据紫外线吸光度估计钴 (II) 结合的 K-2 为 15 +/- 5 μM。对两个突变肽的研究证实了 hdm2 中结合残基的分配，并表明先前通过序列比对提出的 Thr 455 的协调是不正确的。在存在和不存在金属的情况下 hdm2 的结构研究表明，圆二向色光谱仅存在少量二级结构。金属结合似乎并不像其他两种环指蛋白那样使折叠成核。然而，荧光能量转移的距离测量表明，Tyr 489 残基仅距第一个金属中心约 14 埃，这表明 hdm2 蛋白以紧凑的形式存在，至少在金属离子存在的情况下是这样。总之，hdm2 结合金属和 RNA，但 RNA 结合似乎并不以锌依赖性方式发生。
The hdm2 oncoprotein contains a C-terminal domain that binds RNA and has been suggested to bind zinc(II) in an unusual RING finger domain in which Thr 455 was postulated as a ligand. We have reported experiments to test whether this C-terminal cysteine-rich motif is indeed a RING finger domain. We also tested the affinity of the hdm2 C-terminal peptide for metal binding, metal linkage to the folding of the C-terminal peptide, and the peptide's affinity for RNA. Truncation mutants demonstrate that amino acids 425-491 are necessary and sufficient for RNA binding. However, divalent metal ions do not seem to affect the specific RNA recognition. Metal binding studies suggest that hdm2 indeed binds to two molecules of zinc in an intertwined motif similar to the BRCA1 RING finger peptide. However, there is no similarity in overall tertiary structure, nor is there direct sequence homology with other RING fingers. Fluorescence energy transfer studies give a dissociation constant of (0.22 +/- 0.03) mu M for cobalt(II) binding to site 1, while K-2 for cobalt(II) binding was estimated to be 15 +/- 5 mu M from ultraviolet absorbance. Studies of two mutant peptides confirm the assignment of binding residues in hdm2 and suggest that the coordination of Thr 455 previously proposed by sequence alignments is incorrect. Structural studies of hdm2 in the presence and absence of metal indicate only a small amount of secondary structure by circular dichroic spectroscopy. Metal binding did not seem to nucleate folding as in the case of two other RING finger proteins. However, distance measurement from fluorescence energy transfer indicated that the Tyr 489 residue was only similar to 14 Angstrom away from the first metal center, suggesting that the hdm2 protein exists in a compact form, at least in the presence of metal ion. In summary, hdm2 binds metal and RNA, but the RNA binding does not seem to occur in a zinc-dependent manner.