Abstract
Copper (Cu) is an essential trace element but toxic in free form. After cell uptake, Cu is transferred, via direct protein-protein interactions, from the chaperone Atox1 to the Wilson disease protein (WD) for incorporation into Cu-dependent enzymes. Cu binds to a conserved C1XXC2 motif in the chaperone as well as in each of the cytoplasmic metal-binding domains of WD. Here, we dissect mechanism and thermodynamics of Cu transfer from Atox1 to the fourth metal binding domain of WD. Using chromatography and calorimetry together with single Cys-to-Ala variants, we demonstrate that Cu-dependent protein heterocomplexes require the presence of C1 but not C2. Comparison of thermodynamic parameters for mutant versus wild type reactions reveals that the wild type reaction involves strong entropy-enthalpy compensation. This property is explained by a dynamic inter-conversion of Cu-Cys coordinations in the wild type ensemble and may provide functional advantage by protecting against Cu mis-ligation and bypassing enthalpic traps.
Highlights
Found to depend on the metal-loading status as well as on phosphorylation events[10]
Earlier in vitro[16,17,18,19,20,21] and in silico[22] work has shown that Cu+ transfer from Atox[1] to metal-binding domains of Wilson disease protein (WD) and MK proceeds in a two-step process, via Cu-bridged heterodimeric protein complexes (Step 1: formation of an Atox1-Cu-WD complex; Step 2: decomposition of protein heterocomplex into products; Fig. 1) where the metal is shared between the two metal-binding sites
For Atox[1] and WD4, we used size exclusion chromatography (SEC) in combination with titration calorimetry to analyze the reaction in Fig. 126 and we found that Atox1-Cu-WD4 protein heterocomplex formation is driven by favorable enthalpy and entropy changes, whereas the overall reaction, from Atox[1] to WD4, relies on only enthalpy
Summary
Found to depend on the metal-loading status as well as on phosphorylation events[10]. Earlier in vitro[16,17,18,19,20,21] and in silico[22] work has shown that Cu+ transfer from Atox[1] to metal-binding domains of WD and MK proceeds in a two-step process, via Cu-bridged heterodimeric protein complexes (Step 1: formation of an Atox1-Cu-WD complex; Step 2: decomposition of protein heterocomplex into products; Fig. 1) where the metal is shared between the two metal-binding sites. We dissect the Cu+ transport reaction on a molecular level by identifying what Cu-sulfur coordinations are populated in the ensemble of Atox1-Cu-WD4 heterocomplexes, along with a mechanistic explanation based on thermodynamics. We show that Cu+-dependent protein heterocomplexes only form when the 1st Cys in the metal-binding motif of either protein is intact. Thermodynamic analysis of mutant Atox1-Cu-WD4 heterocomplexes points to enthalpy-entropy compensation in the wild type system: this is explained by dynamic interconversion of the two identified tricoordinate Cu+-Cys sites in the wild type protein heterocomplex
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