Multinuclear Metal-Binding Ability of the N-Terminal Region of Human Copper Transporter Ctr1: Dependence Upon pH and Metal Oxidation State

Maria Incoronata Nardella; Mariagrazia Fortino; Alessandra Barbanente; Giovanni Natile; Adriana Pietropaolo; Fabio Arnesano

doi:10.3389/fmolb.2022.897621

Multinuclear Metal-Binding Ability of the N-Terminal Region of Human Copper Transporter Ctr1: Dependence Upon pH and Metal Oxidation State

Front Mol Biosci. 2022 May 5:9:897621. doi: 10.3389/fmolb.2022.897621. eCollection 2022.

Authors

Maria Incoronata Nardella¹, Mariagrazia Fortino², Alessandra Barbanente¹, Giovanni Natile¹, Adriana Pietropaolo², Fabio Arnesano¹

Affiliations

¹ Department of Chemistry, University of Bari Aldo Moro, Bari, Italy.
² Dipartimento di Scienze Della Salute, University of Catanzaro, Catanzaro, Italy.

Abstract

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_1-14 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_1-14-Cu(II), bimetallic Ctr1_1-14-2Cu(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.

Keywords: Ctr1; DFT calculations; NMR specroscopy; copper; mass spectrometry; metal transport; redox processes; silver.