ESI-MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I)

Abstract

Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization-mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750 nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.

Keywords: Cu homeostasis; Cu-thiolate clusters; ESI–MS; Zn homeostasis; metallothionein; phosphorescence.

Graphical Abstract

Species with the same stoichiometry and similar emission wavelengths form in MT1A, MT2, and MT3.

Fig. 1

Comparison of human MT1A (Uniprot #P04731), MT2 (Uniprot #P02795), MT3 (Uniprot #P25713), and MT4 (sequence from Moleirinho et al.) amino acid sequences. Amino acids labeled in red are different compared to the corresponding amino acid in MT1A. Amino acids labeled in blue are similar amino acids to the corresponding amino acid in MT1A.

Fig. 2

ESI–mass spectral data for the titration of Cu(I) into 50.1 μMapo MT2 at pH 7.4. The full MT2 protein is observed at 7325 Da in the mass spectrum. The main species forming after the addition of Cu(I) are Cu₄-MT2, Cu₆-MT2, Cu₁₀-MT2, Cu₁₃-MT2, and Cu₁₅-MT2. A fraction of the protein has had the first “GSM” amino acids cleaved from the protein and is seen at 6795 Da (denoted Apo*). These amino acids are not part of the native protein sequence and do not affect metal binding. The Cu(I) metallation of this cleaved MT2 protein mirrors that of the full MT2 protein and the main species that form are Cu₄*, Cu₆*, Cu₁₀*, Cu₁₃*, and Cu₁₅*. The masses of these species are 531 Da lower than the corresponding species in the full MT2 protein. The metal equivalences noted on the figure refer to the amount of Cu(I) bound to the protein.

Fig. 3

Speciation resulting from Cu(I) addition to apo MT1A, MT2, and MT3. (A) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT1A. Data originally published in Melenbacher et al. (B) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT2. (C) Experimental speciation (symbols) and simulated HySS speciation (lines) for Cu(I) binding to apo MT3. Data originally published in Melenbacher and Stillman. Log K_F values for key species shown in Fig. 6 and Table 3.

Fig. 4

Simulated speciation for two values of log K₉. Solid lines: Overall speciation if log K₉ = 4.93 and log K₁₀ = 29.31. Dashed lines: Overall speciation if log K₉ = 14.09 and log K₁₀ = 19.91. Remaining log K_F values are as reported in Table 3.

Fig. 5

Simulated speciation and mass spectral profiles for varying log K₁₀ values. (A) Simulated speciation where log K₁₀ = 19.91, the value found to best simulate the experimental data in Fig. 3B. (B) Simulated mass spectral profile for the speciation in A with 9.7 mol. eq. Cu(I). (C) Simulated speciation where log K₁₀ʹ = 18.91(i.e. log K₁₀ − 1). (D) Simulated mass spectral profile for the speciation in C with 9.7 mol. eq. Cu(I). (E) Simulated speciation where log K₁₀ʺ = 20.91 (i.e. log K₁₀ + 1). (F) Simulated mass spectral profile for the speciation in E with 9.7 mol. eq. Cu(I).

Fig. 6

Log K_F values for observed species in the Cu(I) titration of MT1A (black squares), MT2 (red circles), and MT3 (blue triangles). Values listed in Table 3.

Fig. 7

Deconvoluted Cu₅Zn₅/Cu₆Zn₄-MT2 peak calculated from the total ion chromatogram averaged over three separate times points.

Fig. 8

Speciation resulting from ⁶³Cu(I) addition to ⁶⁸Zn₇-MT1A (A), ⁶⁸Zn₇-MT2 (B), and ⁶⁸Zn₇-MT3 (C) as determined from the corresponding ESI–mass m/z data (reported in Melenbacher et al. and Melenbacher and Stillman^,). Only key species are shown for clarity. Full speciation shown in Supplementary Fig. S1.

Fig. 9

Abundance of Zn₇-MT (black squares), Cu₁Zn₇-MT (red circles), and Cu₂Zn₆-MT (blue triangles) in MT1A (A), MT2 (B), and MT3 (C). Abundances calculated from ESI–MS data as first reported in Melenbacher et al. and Melenbacher and Stillman.^,

Scheme 1

Proposed pathway of species forming from human Zn₇-MT for MT1A, MT2, and MT3 adapted from the pathway proposed for MT2 by Melenbacher and Stillman.

Fig. 10

Comparison of species abundance for each of MT1A, MT2, and MT3 for the three proposed pathways. Abundance of each of the species detected through ESI–MS as shown in Melenbacher et al. and Melenbacher and Stillman.^, Red numbers indicate the total number of metals bound to the protein. (A–E) Pathway 1 species formed in MT1A (black), MT2 (red), and MT3 (blue). (F–J) Pathway 2 species formed in MT1A (black), MT2 (red), and MT3 (blue). (K) Pathway 3 species formed in MT1A (black), MT2 (red), and MT3 (blue). Isoform-dependent differences in the abundance of Zn₇-MT, Cu₁Zn₇-MT, and Cu₂Zn₆-MT shown in Fig. 9.

Fig. 11

Fraction of different M₁₀ and M₁₂ species forming in MT1A, MT2, and MT3 as a function of Cu(I) bound to the protein. Fractions determined from the ESI–mass spectral data reported in Melenbacher et al. and Melenbacher and Stillman.^, (A) Left: Fraction of Cu₅Zn₅-MT2 (red) and Cu₆Zn₄-MT2 (gray). Right: Fraction of Cu₈Zn₄-MT2 (blue), Cu₉Zn₃-MT2 (cyan), and Cu₁₀Zn₂-MT2 (pink). (B) Left: Fraction of Cu₅Zn₅-MT1A (red) and Cu₆Zn₄-MT1A (gray). Right: Fraction of Cu₈Zn₄-MT1A (blue), Cu₉Zn₃-MT1A (cyan), and Cu₁₀Zn₂-MT1A (pink). (C) Left: Fraction of Cu₅Zn₅-MT3 (red) and Cu₆Zn₄-MT3 (gray). Right: Fraction of Cu₈Zn₄-MT3 (blue), Cu₉Zn₃-MT3 (cyan), and Cu₁₀Zn₂-MT3 (pink).

Fig. 12

ESI–mass spectra and emission spectra of Cu₅Zn₅-MT and Cu₆Zn₄-MT forming following stepwise Cu(I) metallation of Zn₇-MT1A, Zn₇-MT2, and Zn₇-MT3.The x-axis for the mass spectra shows a 60 Da range centered on the mass of Cu₆Zn₄-MT for each of MT1A, MT2, and MT3. (A) ESI–mass spectrum (left) and emission spectrum (right) for 5.0 mol. eq. Cu(I) bound to Zn₇-MT1A. (B) ESI–mass spectrum (left) and emission spectrum (right) for 6.3 mol. eq. Cu(I) bound to Zn₇-MT2. (C) ESI–mass spectrum (left) and emission spectrum (right) for 5.3 mol. eq. Cu(I) bound to Zn₇-MT3. Data originally reported in Melenbacher et al. and Melenbacher and Stillman.^,

Fig. 13

Room temperature emission for the addition of ⁶³Cu(I) to ⁶⁸Zn₇-MT1A/2/3. (A) Emission spectra measured for the addition of ⁶³Cu(I) to ⁶⁸Zn₇-MT2 at pH 7.4. (B) Emission spectra measured for the addition of ⁶³Cu(I) to ⁶⁸Zn₇-MT1A at pH 7.4. (C) Emission spectra measured for the addition of ⁶³Cu(I) to ⁶⁸Zn₇-MT3 at pH 7.8. (D) Normalized emission spectra for MT1A (black), MT2 (red), and MT3 (blue) when 2.9–3.4 mol. eq. Cu(I) is bound to the protein. Data originally reported in Melenbacher et al., and Melenbacher and Stillman.^,