Metal binding and interdomain thermodynamics of mammalian metallothionein-3: enthalpically favoured Cu+ supplants entropically favoured Zn2+ to form Cu4 + clusters under physiological conditions

Abstract

Metallothioneins (MTs) are a ubiquitous class of small metal-binding proteins involved in metal homeostasis and detoxification. While known for their high affinity for d¹⁰ metal ions, there is a surprising dearth of thermodynamic data on metals binding to MTs. In this study, Zn²⁺ and Cu⁺ binding to mammalian metallothionein-3 (MT-3) were quantified at pH 7.4 by isothermal titration calorimetry (ITC). Zn²⁺ binding was measured by chelation titrations of Zn₇MT-3, while Cu⁺ binding was measured by Zn²⁺ displacement from Zn₇MT-3 with competition from glutathione (GSH). Titrations in multiple buffers enabled a detailed analysis that yielded condition-independent values for the association constant (K) and the change in enthalpy (ΔH) and entropy (ΔS) for these metal ions binding to MT-3. Zn²⁺ was also chelated from the individual α and β domains of MT-3 to quantify the thermodynamics of inter-domain interactions in metal binding. Comparative titrations of Zn₇MT-2 with Cu⁺ revealed that both MT isoforms have similar Cu⁺ affinities and binding thermodynamics, indicating that ΔH and ΔS are determined primarily by the conserved Cys residues. Inductively coupled plasma mass spectrometry (ICP-MS) analysis and low temperature luminescence measurements of Cu-replete samples showed that both proteins form two Cu₄ ⁺-thiolate clusters when Cu⁺ displaces Zn²⁺ under physiological conditions. Comparison of the Zn²⁺ and Cu⁺ binding thermodynamics reveal that enthalpically-favoured Cu⁺, which forms Cu₄ ⁺-thiolate clusters, displaces the entropically-favoured Zn²⁺. These results provide a detailed thermodynamic analysis of d¹⁰ metal binding to these thiolate-rich proteins and quantitative support for, as well as molecular insight into, the role that MT-3 plays in the neuronal chemistry of copper.

Fig. 1. Structures of the N-terminal β-domain (blue, PDB: 1MHU), containing 3 Cd²⁺ ions bound to 9 cysteine residues, and the C-terminal α-domain (tan, PDB: 2MHU), containing 4 Cd²⁺ ions bound to 11 cysteine residues, determined by NMR measurements, of the human MT-2 protein.

Fig. 2. Representative thermograms for DTPA chelation of Zn²⁺ from Zn₇MT-3 in 100 mM buffer and 150 mM NaCl at pH 7.4; data for the second event are masked (red) and data for the first event were fitted (solid line) to a one-site binding mode with the best-fit values and fit errors: (A) HEPES: n_ITC = 6.93 ± 0.05, K_ITC = 1.2 (±0.3) × 10⁷ and ΔH°_ITC = −9.64 ± 0.09 kcal mol⁻¹; (B) Bis–Tris: n_ITC = 6.56 ± 0.01, K_ITC = 5.6 ± 0.3 × 10⁶ and ΔH°_ITC = −11.16 ± 0.04 kcal mol⁻¹; (C) TAPSO: n_ITC = 7.02 ± 0.04, K_ITC = 3.9 (±0.5) × 10⁶ and ΔH°_ITC = −14.55 ± 0.07 kcal mol⁻¹.

Fig. 3. Representative thermograms of the DTPA chelation of Zn²⁺ in 100 mM TAPSO buffer and 150 mM NaCl at pH 7.4; data for the second event are masked (red) and data for the first event were fitted (solid line) to a one-site binding model with best-fit values and fit errors: (A) Zn₄αMT-3: n_ITC = 4.39 ± 0.02, K_ITC = 6.7 (±0.9) × 10⁶ and ΔH°_ITC = −14.19 ± 0.06 kcal mol⁻¹; (B) Zn₃βMT-3: n_ITC = 3.07 ± 0.04, K_ITC = 4.1 (±0.9) × 10⁶ and ΔH°_ITC = −13.8 ± 0.1 kcal mol⁻¹.

Fig. 4. Representative thermograms of Cu⁺ titrated into (A) Zn₇MT-3 and (B) Zn₇MT-2 in 100 mM buffer, 150 mM NaCl and 5 mM GSH at pH 7.4; the data were fitted (solid line) to a one-site binding model with the best-fit values and fit errors for Zn₇MT-3 in Bis–Tris buffer: n_ITC = 6.5 ± 0.2, K_ITC = 6 (±1) × 10⁵ and ΔH°_ITC = 6.5 ± 0.3 kcal mol⁻¹, and for Zn₇MT-2 in MOPS buffer are n_ITC = 8.9 ± 0.1, K_ITC = 1.4 (±0.2) × 10⁶ and ΔH°_ITC = 4.8 ± 0.1 kcal mol⁻¹; insets show enlargements of representative injection peaks, which plot μcal s⁻¹vs. time.

Fig. 5. 77 K luminescence emission spectra of the MT-3 (a) and MT-2 (b) fractions generated upon incubation of Zn₇MT-3 or Zn₇MT-2 (10 μM) with Cu⁺ (200 μM) in the presence of excess GSH (20 mM), followed by MT separation by SEC; insets are the emission decay and lifetimes (τ) at 425 nm and 575 nm obtained by fitting the data with a single decay exponential function.