Nardella Maria Incoronata, Fortino Mariagrazia, Barbanente Alessandra, Natile Giovanni, Pietropaolo Adriana, Arnesano Fabio
Department of Chemistry, University of Bari Aldo Moro, Bari, Italy.
Dipartimento di Scienze Della Salute, University of Catanzaro, Catanzaro, Italy.
Front Mol Biosci. 2022 May 5;9:897621. doi: 10.3389/fmolb.2022.897621. eCollection 2022.
The 14mer peptide corresponding to the N-terminal region of human copper transporter Ctr1 was used to investigate the intricate mechanism of metal binding to this plasma membrane permease responsible for copper import in eukaryotic cells. The peptide contains a high-affinity ATCUN Cu(II)/Ni(II)-selective motif, a methionine-only MxMxxM Cu(I)/Ag(I)-selective motif and a double histidine HH(M) motif, which can bind both Cu(II) and Cu(I)/Ag(I) ions. Using a combination of NMR spectroscopy and electrospray mass spectrometry, clear evidence was gained that the Ctr1 peptide, at neutral pH, can bind one or two metal ions in the same or different oxidation states. Addition of ascorbate to a neutral solution containing Ctr1 and Cu(II) in 1:1 ratio does not cause an appreciable reduction of Cu(II) to Cu(I), which is indicative of a tight binding of Cu(II) to the ATCUN motif. However, by lowering the pH to 3.5, the Cu(II) ion detaches from the peptide and becomes susceptible to reduction to Cu(I) by ascorbate. It is noteworthy that at low pH, unlike Cu(II), Cu(I) stably binds to methionines of the peptide. This redox reaction could take place in the lumen of acidic organelles after Ctr1 internalization. Unlike Ctr1-Cu(II), bimetallic Ctr1-2Cu(II) is susceptible to partial reduction by ascorbate at neutral pH, which is indicative of a lower binding affinity of the second Cu(II) ion. The reduced copper remains bound to the peptide, most likely to the HH(M) motif. By lowering the pH to 3.5, Cu(I) shifts from HH(M) to methionine-only coordination, an indication that only the pH-insensitive methionine motif is competent for metal binding at low pH. The easy interconversion of monovalent cations between different coordination modes was supported by DFT calculations.
对应于人铜转运蛋白Ctr1 N端区域的14聚体肽被用于研究金属与这种负责真核细胞中铜导入的质膜通透酶结合的复杂机制。该肽包含一个高亲和力的ATCUN Cu(II)/Ni(II)选择性基序、一个仅含甲硫氨酸的MxMxxM Cu(I)/Ag(I)选择性基序和一个双组氨酸HH(M)基序，它们都能结合Cu(II)和Cu(I)/Ag(I)离子。通过结合使用核磁共振光谱和电喷雾质谱，有明确证据表明，在中性pH条件下，Ctr1肽能结合一个或两个处于相同或不同氧化态的金属离子。向含有等摩尔比Ctr1和Cu(II)的中性溶液中添加抗坏血酸，不会使Cu(II)明显还原为Cu(I)，这表明Cu(II)与ATCUN基序紧密结合。然而，将pH降至3.5时，Cu(II)离子会从肽上脱离，并容易被抗坏血酸还原为Cu(I)。值得注意的是，在低pH下，与Cu(II)不同，Cu(I)能稳定地结合到肽的甲硫氨酸上。这种氧化还原反应可能在Ctr1内化后发生在酸性细胞器的腔内。与Ctr1-Cu(II)不同，双金属的Ctr1-2Cu(II)在中性pH下易被抗坏血酸部分还原，这表明第二个Cu(II)离子的结合亲和力较低。还原后的铜仍与肽结合，最有可能结合到HH(M)基序上。将pH降至3.5时，Cu(I)从HH(M)基序转移到仅含甲硫氨酸的配位模式，这表明只有对pH不敏感的甲硫氨酸基序在低pH下能够结合金属。密度泛函理论计算支持了单价阳离子在不同配位模式之间的轻松相互转化。
