Lai Z, Freedman D A, Levine A J, McLendon G L
Departments of Chemistry and Molecular Biology, Princeton University, Princeton, New Jersey 08544, USA.
Biochemistry. 1998 Dec 1;37(48):7005-15. doi: 10.1021/bi980596r.
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) microM for cobalt(II) binding to site 1, while K2 for cobalt(II) binding was estimated to be 15 +/- 5 microM 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 approximately 14 A 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.
HDM2癌蛋白含有一个能结合RNA的C末端结构域，并且有人提出它在一个不寻常的RING指结构域中结合锌（II），其中苏氨酸455被假定为配体。我们已经报道了实验来测试这个富含半胱氨酸的C末端基序是否确实是一个RING指结构域。我们还测试了HDM2 C末端肽与金属结合的亲和力、金属与C末端肽折叠的联系以及该肽与RNA的亲和力。截短突变体表明，氨基酸425 - 491对于RNA结合是必要且充分的。然而，二价金属离子似乎并不影响特异性RNA识别。金属结合研究表明，HDM2确实以类似于BRCA1 RING指肽的交织基序结合两个锌分子。然而，其整体三级结构没有相似性，与其他RING指结构也没有直接的序列同源性。荧光能量转移研究得出钴（II）结合到位点1的解离常数为（0.22±0.03）μM，而从紫外吸收估计钴（II）结合的K2为15±5μM。对两个突变肽的研究证实了HDM2中结合残基的归属，并表明之前通过序列比对提出的苏氨酸455的配位是不正确的。在有金属和无金属情况下对HDM2的结构研究通过圆二色光谱表明只有少量二级结构。与其他两种RING指蛋白的情况不同，金属结合似乎并没有引发折叠。然而，荧光能量转移的距离测量表明，酪氨酸残基489距离第一个金属中心仅约14埃，这表明HDM2蛋白至少在存在金属离子的情况下以紧密形式存在。总之，HDM2结合金属和RNA，但RNA结合似乎不是以锌依赖的方式发生。
