Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Abstract

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K a ) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are ϳ10 5 to 10 6 M ؊1 , suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K a values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.

Yeast Cox17 Solution Structure and Copper(I) Binding

“…The K a value for Cox17 is on the order of 10 6 -10 7 M Ϫ1 , roughly similar to the value of ϳ10 5 M Ϫ1 measured by ITC for the human copper chaperone Atox1 (34). This affinity is moderate compared with values obtained for Cu(II) binding to metalloenzymes or Cu(I) binding to metallothionein (34) and is consistent with a role for Cox17 in copper transfer.…”

“…This affinity is moderate compared with values obtained for Cu(II) binding to metalloenzymes or Cu(I) binding to metallothionein (34) and is consistent with a role for Cox17 in copper transfer. The cytosol is proposed to contain extremely limited free copper (39), suggesting that Atox1 retains its copper in the presence of other higher affinity binding sites via a slow kinetic rate constant k off .…”

“…Because Cox17 functions within the mitochondrion, which contains an accessible pool of copper that is not associated with specific proteins (16), retaining copper may not pose the same problem. For the Atox1 pathway, the similar Cu(I) affinities of the chaperone and its target domains in the Wilson disease protein suggest that copper exchange is under kinetic, rather than thermodynamic, control (34). The situation may be the same for copper transfer from Cox17 to Sco1 and Cox11, but the Cu(I) binding affinities of these two target proteins have not yet been determined.…”

“…Binding of Cu(I) by Cox17-ITC data for Cu(I) binding to Cox17 were obtained using protocols developed in our laboratory specifically for analysis of Cu(I) binding to cysteine residues (34). The validity of this approach was tested by titrating Mg(II) into Cox17 (Fig.…”

“…After subtraction of this heat of dilution, a nonlinear least squares method was used to obtain the best fit parameters for the number of binding sites, n, the association constant, K a , and the change in enthalpy ⌬H°. Two negative control experiments were used to address the specificity of copper binding (34). First, Mg(II) was titrated into 20 M Cox17 using 300 M MgCl 2 prepared in 10 mM HCl and 1 M NaCl.…”