Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109, United States.
Program in Biophysics, University of Michigan, Ann Arbor, Michigan 48109, United States.
Inorg Chem. 2022 Mar 28;61(12):5084-5091. doi: 10.1021/acs.inorgchem.2c00057. Epub 2022 Mar 14.
Long interspersed nuclear elements-1 (L1) are autonomous retrotransposons that encode two proteins in different open reading frames (ORF1 and ORF2). The ORF1p, which may be an RNA binding and chaperone protein, contains a three-stranded coiled coil (3SCC) domain that facilitates the formation of the biologically active homotrimer. This 3SCC domain is composed of seven amino acid (heptad) repeats as found in native and designed peptides and a stammer that modifies the helical structure. Cysteine residues occur at three hydrophobic positions (2 and 1 sites) within this domain. We recently showed that the cysteine layers in ORF1p and model designed peptides bind the toxic metalloid lead(II) with high affinities, a feature that had not been previously recognized. However, there is little understanding of how essential metal ions might interact with this metal binding domain. We have, therefore, investigated the copper(I) binding properties of analogous designed 3SCCs that contain cysteine layers within the hydrophobic core. The results from UV-visible and X-ray absorption spectroscopy show that these designed peptides bind Cu(I) with high affinity in a pH-dependent manner. At pH 9, monomeric trigonal planar Cu(I)S centers are formed with 1 equiv of metal, while dinuclear centers form with a second equivalent of metal. At physiologic pH conditions, the dinuclear center forms cooperatively. These data suggest that ORF1p is capable of binding two copper ions to its tris(cysteine) layers. This has major implications for ORF1p coiled coil domain stability and dynamics, ultimately potentially impacting the resulting biological activity.
长散在核元件-1（L1）是自主的反转录转座子，在不同的开放阅读框（ORF1 和 ORF2）中编码两种蛋白质。ORF1p 可能是一种 RNA 结合和伴侣蛋白，含有一个三股螺旋卷曲（3SCC）结构域，有助于形成具有生物活性的同源三聚体。该 3SCC 结构域由在天然和设计的肽中发现的七个氨基酸（七肽）重复序列和一个改变螺旋结构的口吃组成。半胱氨酸残基出现在该结构域的三个疏水区（2 位和 1 位）。我们最近表明，ORF1p 和模型设计肽中的半胱氨酸层以高亲和力结合有毒类金属铅（II），这一特征以前尚未被认识到。然而，对于必需金属离子如何与这个金属结合域相互作用，我们知之甚少。因此，我们研究了类似的设计 3SCC 中的铜（I）结合特性，这些设计的肽在疏水区内含有半胱氨酸层。紫外可见和 X 射线吸收光谱的结果表明，这些设计的肽以 pH 依赖性的方式以高亲和力结合 Cu（I）。在 pH 9 下，形成单体三角平面 Cu（I）S 中心，与 1 当量的金属结合，而双核中心与第二个当量的金属结合。在生理 pH 条件下，双核中心协同形成。这些数据表明，ORF1p 能够将两个铜离子结合到其三（半胱氨酸）层上。这对 ORF1p 卷曲螺旋结构域的稳定性和动力学有重大影响，最终可能影响到生物活性。
