Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme
Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme
复制标题
锤头核酶保守 A9 金属位点中 Mn2 和 Cd2 与硫代磷酸酯配位的光谱表征
DOI：
10.1016/j.jinorgbio.2022.111754
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发表时间：
2022
影响因子：
3.9
通讯作者：
DeRose, Victoria J.
中科院分区：
文献类型：
--
作者：
Hunsicker-Wang, Laura M.;Vogt, Matthew J.;Hoogstraten, Charles G.;Cosper, Nathaniel J.;Davenport, Audrey M.;Hendon, Christopher H.;Scott, Robert A.;Britt, R. David;DeRose, Victoria J.
硫代磷酸酯修饰在核酸领域中具有广泛的用途。由于硫取代氧可以改变金属配位偏好，硫代磷酸酯金属拯救实验是鉴定影响大RNA中特定性质的金属配位位点的有力方法。锤头状核酶（HHRz）的A9/G10.1金属结合位点先前已通过硫代磷酸酯拯救实验显示出功能上重要。虽然A9-SRp取代在Mg 2+中是抑制性的，但亲硫Cd 2+拯救了HHRz活性。Mn 2+也经常用于硫代磷酸金属拯救研究，但不支持A9-SRpHHRz的活性。在这里，我们使用EPR，电子自旋回波包络调制（ESEEM），和X-射线吸收光谱方法直接探测结构的后果Mn 2+和Cd 2+协调RpandSp硫代磷酸修饰在A9/G10.1网站的截断锤头状核酶（tHHRz）。结果表明，虽然Cd 2+确实与硫代取代配体中的S结合，但Mn 2+与该硫代磷酸酯的非硫氧代基团配位，无论异构体如何。计算模型表明，在金属-二甲基硫代磷酸盐模型中，Mn单键O配位比Mn单键S配位具有能量优先性。在tHHRz的情况下，所得的Mn 2+配位优先的氧在eitherRporSpA 9硫代磷酸酯差异调谐催化活性，Mn单键O配位在A9-SRp硫代磷酸酯酶是抑制性的。
Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-SRpsubstitution is inhibitory in Mg2+, thiophilic Cd2+rescues HHRz activity. Mn2+is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-SRpHHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn2+and Cd2+coordination toRpandSpphosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd2+does indeed bind to S in the thio-substituted ligand, Mn2+coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of Mnsingle bondO over Mnsingle bondS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn2+coordination preference of oxygen in eitherRporSpA9 phosphorothioates differentially tunes catalytic activity, with Mnsingle bondO coordination in the A9-SRpphosphorothioate enzyme being inhibitory.