Model peptides provide new insights into the role of histidine residues as potential ligands in human cellular copper acquisition via Ctr1

Abstract

Cellular acquisition of copper in eukaryotes is primarily accomplished through the Ctr family of copper transport proteins. In both humans and yeast, methionine-rich "Mets" motifs in the amino-terminal extracellular domain of Ctr1 are thought to be responsible for recruitment of copper at the cell surface. Unlike yeast, mammalian Ctr1 also contains extracellular histidine-rich motifs, although a role for these regions in copper uptake has not been explored in detail. Herein, synthetic model peptides containing the first 14 residues of the extracellular domain of human Ctr1 (MDHSHHMGMSYMDS) have been prepared and evaluated for their apparent binding affinity to both Cu(I) and Cu(II). These studies reveal a high affinity Cu(II) binding site (log K = 11.0 ± 0.3 at pH 7.4) at the amino-terminus of the peptide as well as a high affinity Cu(I) site (log K = 10.2 ± 0.2 at pH 7.4) that utilizes adjacent HH residues along with an additional His or Met ligand. These model studies suggest that the histidine domains may play a direct role in copper acquisition from serum copper-binding proteins and in facilitating the reduction of Cu(II) to the active Ctr1 substrate, Cu(I). We tested this hypothesis by expressing a Ctr1 mutant lacking only extracellular histidine residues in Ctr1-knockout mouse embryonic fibroblasts. Results from live cell studies support the hypothesis that extracellular amino-terminal His residues directly participate in the copper transport function of Ctr1.

Figure 1

Human Ctr1 assembles as a homotrimer in the plasma membrane with an amino-terminal extracellular domain equipped with His (H) and Met (M) potential metal-binding residues. All extracellular His and Met residues are indicated. The functionally critical TMD2 methionine residues and carboxyl-terminal cysteine and histidine residues are indicated.

Figure 2

Absorbance spectra of Ctr1 model peptides (~500 μM) with 1 equivalent of Cu(II) at pH 7.4 in HEPES buffer. Peptides with His in the third position from the amino-terminus display absorbance at 525 nm characteristic of a typical ATCUN Cu(II) binding site. Substitution of all His-to-Ala, individual substitution of the His at position 3 (H3A) or acetyl capping of the N-terminus (Ac-Ctr1-14) results in a different type of binding site and a shift in λ_max compared to the wild-type peptide (Ctr1-14).

Figure 3

Visible spectra showing the results of a titration of NTA into a 500 μM solution of 1:1 P:Cu(II) in 50 mM HEPES buffer at pH 7.4, where P = Ctr1-14.

Figure 4

Spectra from a titration of 50 equivalents of Ctr1-14 peptide into 75 μM BCA and 25 μM [Cu(I)(CH₃CN)₄]PF₆ in 50 mM HEPES buffer at pH 7.4 and 0.5% CH₃CN.

Figure 5

Ascorbate-dependent reduction of Cu(II) in complex with Ctr1-14 model peptides containing the ATCUN site. Solutions of 500 μM peptide-Cu(II) complex and 1 mM ascorbate in 50 mM HEPES at pH 7.4 monitored for 1 hour with UV-Vis at 525 nm, the characteristic absorbance band due to the Cu(II) complex.

Figure 6

Immunofluorescent imaging of Ctr1 expression in various cell lines. Fixed cells were probed with specific primary antibody for Ctr1 and secondary antibody fused to a green fluorescent dye. Slides were mounted with DAPI stain to visualize the nucleus (blue). a) MEF Ctr1^−/− cells with both alleles of Ctr1 deleted do not express Ctr1 and show only low background fluorescence. b) MEF Ctr1^−/− cell line stably expressing Ctr1. General distribution of green fluorescence is typical for normally-functioning Ctr1, which localizes to the plasma membrane as well as endosomes. c) MEF Ctr1^−/− cell line stably expressing Ctr1ntHA mutant shows typical distribution expected for Ctr1 localized to the plasma membrane and endosomes.

Figure 7

High CCS levels in Ctr1ntHA expressing cells indicate a copper deficient phenotype. a) Immunoblot showing expression levels of proteins recognized by specific antibodies against Ctr1, CCS and actin in Ctr1 knockout MEF cells (Ctr1^−/−), Ctr1^−/− cells stably expressing transgenic human wild type Ctr1 (Ctr1) or the amino-terminal Ctr1 His to Ala mutant (Ctr1ntHA). Cells expressing Ctr1ntHA have high CCS levels compared to the Ctr1 transgenic control. High CCS levels in cells expressing Ctr1ntHA indicate that His mutation causes an intracellular copper deficiency. b) CCS levels quantified over three experiments using an individual stable cell line expressing Ctr1ntHA. Data were quantified using Photoshop software. Band intensities from CCS were normalized to band intensities from an actin loading control. Data from separate experiments were compared by normalizing all data to the relative intensity of CCS expression levels of Ctr1^−/− cells from each experiment.