Non-coordinative metal selectivity bias in human metallothioneins metal-thiolate clusters

Abstract

Mammalian metallothioneins (MT-1 through MT-4) are a class of metal binding proteins containing two metal-thiolate clusters formed through the preferential coordination of d10 metals, Cu(i) and Zn(ii), by 20 conserved cysteine residues located in two protein domains. MT metalation (homometallic or heterometallic Zn(ii)/Cu(i) species) appears to be isoform specific and controlling zinc and copper concentrations to perform specific and distinct biological functions. Structural and functional relationships, and in vivo metalation studies, identified evolutionary features defining the metal-selectivity nature for MTs. Metallothionein-3 (MT-3) has been shown to possess the most pronounced Cu-thionein character forming Cu(i)-containing species more favorably than metallothionein-2 (MT-2), which possesses the strongest Zn-thionein character. In this work, we identify isoform-specific determinants which control metal binding selectivity bias in different MTs isoforms. By studying the reactivity of Zn7MT-2, Zn7MT-3 and Zn7MT-3 mutants towards Cu(ii) to form Cu(i)4Zn4MTs, we have identified isoform-specific key non-coordinating residues governing folding/outer sphere control of metal selectivity bias in MTs metal clusters. By mutating selected residues and motifs in MT-3 to the corresponding MT-2 amino acids, we dissected key roles in modulating cluster dynamic and metal exchange rates, in increasing the Cu(i)-affinity in MT-3 N-terminal β-domain and/or modulating the higher stability of the Zn(ii)-thiolate cluster in MT-2 β-domain. We thus engineered MT-3 variants in which the copper-thionein character is converted into a zinc-thionein. These results provide new insights into the molecular determinants governing metal selectivity in metal-thiolate clusters.

Figure 1.

(A) Schematic representation of the reactivity of Zn₇MTs towards Cu(II) and products of the reactions investigated in this study. (B) Amino acid alignment of human MT-2A and MT-3 sequences. Conserved metal coordinating cysteines residues are highlighted in yellow and amino acid positions in MT-3 sequence that have been mutated in this work to the corresponding ones in MT-2 are highlighted in light green. An amino acid conservation plot between the two sequences is presented (the scores indicate the degree of similarity between two corresponding position; scheme generated with Jalview).

Figure 2.

Electronic absorption spectra of 5 µM Zn₇MT-3 and 5 µM Cu(I)₄Zn₄MT-3 (A), and 5 µM Zn₇MT-2 and 5 µM Cu(I)₄Zn₄MT-2 (B) in 25 mM Tris-HCl/50 mM NaCl, pH 8.0.

Figure 3.

(A) Luminescence emission spectra for Cu(I)₄Zn₄MT-2 (red) and Cu(I)₄Zn₄MT-3 (black) obtained upon reaction of 10 µM Zn₇MT-2 with 4 Cu²⁺ equivalents in 25 mM Tris/HCl, 50 mM NaCl, pH 8, recorded on frozen samples at 77 K upon excitation at 320 nm. (B) Lifetime determination of the emissive bands at 425 nm and 575 nm in Cu(I)₄Zn₄MT-2 and corresponding fits obtained with a single exponential decay function.

Figure 4.

Size-exclusion chromatograms obtained upon the reaction of 5 µM Zn₇MT-2 or Zn₇MT-3 with 0, 4, or 8 Cu²⁺ equivalents incubated aerobically for 1 h or 24 h in 25 mM Tris/HCl, 50 mM NaCl, pH 8.0, recorded at 220 nm (to monitor Zn(II) clusters stability) and 260 nm (to monitor Cu(I) cluster stability).

Figure 5.

Time-dependent kinetic UV absorption spectra of the product of the reaction between 2.5 µM Zn₇MT-3 in 25 mM Tris-HCl/50 mM NaCl, pH 8.0 and 4 Cu²⁺ equivalents followed for 600 s at 25 °C (A) and the kinetic traces at 220 nm and 260 nm monitoring Zn(II) release and Cu(I) binding (B) in MT-3.

Figure 6.

Kinetic profile of the reaction of 2.5 µM Zn₇MT-2 or Zn₇MT-3 in 25 mM Tris/HCl, 50 mM NaCl, pH 8.0 with 4 Cu²⁺ equivalents monitored at 260 nm (MT-3, red; MT-2 blue) and at 220 nm (MT-3, black; MT-2 gray) for 600 s at 25 °C. The data reveal that Cu(I) binding reaction is > 95% completed after approx. 75 s for MT-3 and 250 s for MT-2.

Figure 7:

Sequence alignment of MT-3 mutants investigated in this work. Amino acids in MT-3 sequence mutated to the corresponding amino acids present in MT-2 sequence are highlighted in light green, while deletions are highlighted in red boxes.

Figure 8.

Luminescence emission spectra for Cu(I)₄Zn₄MTs obtained from the reaction of 10 µM Zn₇MT samples in 25 mM Tris/HCl, 50 mM NaCl, pH 8.0 with 4 Cu²⁺ equivalents, recorded on frozen samples at 77 K upon excitation at 320 nm (a: MT-3; b: ΔT5 MT-3; c: P7SP9A MT-3, d: ΔT5-P7SP9A MT-3; e: E23K MT-3; f: E23KG24E MT-3; g: Δ55–60 MT-3; h: ΔT5-P7SP9A-E23K MT-3, i: ΔT5-P7SP9A-Δ55–60 MT-3; j: ΔT5-P7SP9A-E23K-Δ55–60 MT-3; k: ΔT5-P7SP9A-E23KE24K-Δ55–60 MT-3; and l: MT-2).

Figure 9.

Kinetic traces of the reaction of 2.5 µM Zn₇MT-2, Zn₇MT-3, or Zn₇MT-3 mutants in 25 mM Tris/HCl, 50 mM NaCl, pH 8.0 with 4 Cu²⁺ equivalents monitored at 260 nm at 25 °C.