A Deeper Insight in Metal Binding to the hCtr1 N-terminus Fragment: Affinity, Speciation and Binding Mode of Binuclear Cu2+ and Mononuclear Ag+ Complex Species

Abstract

Ctr1 regulates copper uptake and its intracellular distribution. The first 14 amino acid sequence of the Ctr1 ectodomain Ctr1_(1-14) encompasses the characteristic Amino Terminal Cu²⁺ and Ni²⁺ binding motif (ATCUN) as well as the bis-His binding motif (His5 and His6). We report a combined thermodynamic and spectroscopic (UV-vis, CD, EPR) study dealing with the formation of Cu²⁺ homobinuclear complexes with Ctr1_(1-14), the percentage of which is not negligible even in the presence of a small Cu²⁺ excess and clearly prevails at a M/L ratio of 1.9. Ascorbate fails to reduce Cu²⁺ when bound to the ATCUN motif, while it reduces Cu²⁺ when bound to the His5-His6 motif involved in the formation of binuclear species. The histidine diade characterizes the second binding site and is thought to be responsible for ascorbate oxidation. Binding constants and speciation of Ag⁺ complexes with Ctr1_(1-14), which are assumed to mimic Cu⁺ interaction with N-terminus of Ctr1_(1-14), were also determined. A preliminary immunoblot assay evidences that the anti-Ctr1 extracellular antibody recognizes Ctr1_(1-14) in a different way from the longer Ctr1_(1-25) that encompasses a second His and Met rich domain.

Keywords: Ctr1; affinity; antibody recognition; copper; model transporter; reductase; silver; speciation.

Figure 1

Species distribution for the Cu²⁺- Ctr1_(1-14) system. C_Ctr1 = 5 × 10⁻⁴ mol dm⁻³; Cu²⁺/Ctr1_(1-14) concentration ratio is 0.9, 1.2, 1.6 and 1.9 for (a–d), respectively.

Figure 2

Total concentration of Cu²⁺-Ctr1_(1-14) mononuclear and binuclear complexes species at Cu²⁺/Ctr1_(1-14) concentration ratios equal to 0.9, 1.2, 1.6 and 1.9 for (a–d), respectively.

Figure 3

(a) Ascorbate-dependent reduction of Cu²⁺ in the presence or absence of Ctr1_(1-14) at different metal/peptide ratios. Peptide (25 μM) and ascorbate (100 μM) solutions in MOPS (10 mM) at pH 7.2 were monitored for 30 min by UV-vis spectra at 265 nm. Change of ascorbate absorbance band: ascorbate without (black line) and with Cu²⁺ (red line), 1:1 Cu²⁺/Ctr1_(1-14) ratio (blue), 2:1 Cu²⁺/Ctr1_(1-14) ratio (magenta), 3:1 Cu²⁺/Ctr1_(1-14) ratio (green). (b) UV-vis spectra of Cu²⁺-Ctr1_(1-14) system carried out in the same experimental condition of Cu²⁺ ascorbate-dependent reduction; the brown line represents the difference spectra.

Figure 4

Species distribution for the Cu²⁺-Ctr1_(1-14) system. C_Ctr1 = 2.5 × 10⁻⁵ mol dm⁻³; Cu²⁺/Ctr1_(1-14) concentration ratio is 1.9.

Figure 5

Species distribution of Ag⁺ complexes with Ctr1_(1-14) (L). Ag⁺/Ctr1_(1-14) = 0.9; the analytical concentration of Ctr1_(1-14) is 5 × 10⁻⁴ mol dm⁻³.

Figure 6

ESR spectra (150 K) of the complex species of Ctr1_(1-14), with Cu²⁺ as function of the pH and the ligand-to-metal ratio (left panel, L:Cu = 1:1; right panel, L:Cu = 1:2). Computed spectra are shown in red.

Figure 7

Immunoblots of Ctr1_(1-14) and Ctr1_(1-25) peptides (1 mM) pre-incubated with different concentrations of copper (L/Cu²⁺ ratios 1:1, 1:2, 1:3) with or without 1% SDS. The membrane was incubated with anti-Ctr1 extracellular domain antibody.