In Vitro Thermodynamic Dissection of Human Copper Transfer from Chaperone to Target Protein

Abstract

Transient protein-protein and protein-ligand interactions are fundamental components of biological activity. To understand biological activity, not only the structures of the involved proteins are important but also the energetics of the individual steps of a reaction. Here we use in vitro biophysical methods to deduce thermodynamic parameters of copper (Cu) transfer from the human copper chaperone Atox1 to the fourth metal-binding domain of the Wilson disease protein (WD4). Atox1 and WD4 have the same fold (ferredoxin-like fold) and Cu-binding site (two surface exposed cysteine residues) and thus it is not clear what drives metal transfer from one protein to the other. Cu transfer is a two-step reaction involving a metal-dependent ternary complex in which the metal is coordinated by cysteines from both proteins (i.e., Atox1-Cu-WD4). We employ size exclusion chromatography to estimate individual equilibrium constants for the two steps. This information together with calorimetric titration data are used to reveal enthalpic and entropic contributions of each step in the transfer process. Upon combining the equilibrium constants for both steps, a metal exchange factor (from Atox1 to WD4) of 10 is calculated, governed by a negative net enthalpy change of ∼10 kJ/mol. Thus, small variations in interaction energies, not always obvious upon comparing protein structures alone, may fuel vectorial metal transfer.

Human cytoplasmic copper chaperones Atox1 and CCS exchange copper ions in vitro

“…Cu transfer from Atox1 to metal-binding domains of the Wilson disease and Menkes disease proteins proceeds via (stable or transient) copper-bridged hetero-dimer complexes where the metal is shared between the two proteins' metal-binding site cysteines in tri-coordinate arrangements (Banci et al 2005;Achila et al 2006;Banci et al 2008Banci et al , 2009). In agreement with NMR data (Banci et al 2009), we showed earlier that upon mixing of Cu-Atox1 and the fourth metal-binding domain of Wilson disease protein (WD4), a stable ternary complex is formed in equilibrium with both substrates and products (Niemiec et al 2012), whereas mixing of CuAtox1 with a two-domain construct of metal-binding domains 5 and 6 of the Wilson disease protein (WD56) resulted in Cu transfer but no detectable heterocomplex (Nilsson et al 2013).…”

“…We found that Cu binding to CCS 1 , like to Atox1 (Niemiec et al 2012), can be probed via changes in the 254 nm/280 nm absorbance ratio since Cu binding to the thiols in the Cys side chains results in increased 254 nm absorbance while the 280 nm absorbance remains constant. Cu titrations to CCS 1 -probed by 254/280 nm absorption ratio of the CCS 1 peak after gel filtration separation (to remove reducing agent that may affect the absorption)-show that Cu binds stoichiometrically forming a 1-to-1 complex with CCS 1 at these conditions (Fig.…”

“…The protein concentration was determined by absorption at 280 nm (e 280 = 5625 cm -1 M -1 ). Atox1 was prepared as previously reported (Niemiec et al 2012). The Cys-to-Ala variant Atox1 (C12A) was also ordered from GenScript and purified like the WT protein.…”

“…The selected excess concentration of the reducing agent DTT (5-7 fold over protein) has been tested out previously to be high enough to keep the Cu ions reduced prior to protein binding but still low enough to allow for stoichiometric Cu 1? loading of the proteins (Niemiec et al 2012(Niemiec et al , 2014. Protein concentrations were determined via extinction coefficients at 280 nm based on amino acid sequences.…”

“…Here, we demonstrate Cu transfer between CCS 1 (and full-length CCS) and Atox1, and vice versa, using an experimental approach that is based on a combination of size exclusion chromatography (SEC) and dual-wavelength absorption detection (Niemiec et al 2012). Because CCS 1 elutes differently from Atox1 in Biometals SEC (although monomeric as shown by NMR), and since the 254/280 nm absorption ratio depends on Cu loading of each protein, we may directly investigate Cu transfer between the proteins via dual-wavelength absorption-detected SEC.…”