title: 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.
authors.affiliation: Chemistry Department, Chemical Biological Center, Umeå University, Umeå, Sweden.
authors.forename: Moritz S
authors.initials: MS
authors.lastname: Niemiec
authors.forename: Christoph F
authors.initials: CF
authors.lastname: Weise
authors.forename: Pernilla
authors.initials: P
authors.lastname: Wittung-Stafshede
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002460:Cell Line; D003300:Copper / Q000378:metabolism*; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D006801:Humans; D056511:Metallochaperones; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: C474500:ATOX1 protein, human; D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D056511:Metallochaperones; D018832:Molecular Chaperones; D003300:Copper; D000251:Adenosine Triphosphatases; C081184:ATP7A protein, human; D000073840:Copper-Transporting ATPases