N-terminal domains of human copper-transporting adenosine triphosphatases (the Wilson's and Menkes disease proteins) bind copper selectively in vivo and in vitro with stoichiometry of one copper per metal-binding repeat.

Jack H. Kaplan,Conrad T. Gilliam,Svetlana Lutsenko,Konstantin Petrukhin,Matthew J. Cooper

doi:10.1074/jbc.272.30.18939

Abstract

N-terminal domains of the Wilson's and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized. Both N-MNK and N-WND bind copper specifically as indicated by the results of metal-chelate chromatography, direct copper-binding measurements, and chemical modification of Cys residues in the presence of different heavy metals. When E. coli cells are grown in the presence of copper, N-MNK and N-WND bind copper in vivo with stoichiometry of 5-6 nmol of copper/nmol of protein. Copper released from the copper-N-MNK and copper-N-WND complexes reacts with the Cu(I)-selective chelator bicinchoninic acid in the absence of reducing agents. This suggests that in proteins, it is bound in reduced Cu(I) form, in agreement with the spectroscopic properties of the copper-bound domains. Copper bound to the domains in vivo or in vitro specifically protects the N-MNK and N-WND against labeling with the cysteine-directed probe; this indicates that Cys residues in the repetitive motifs GMTCXXCXXXIE are involved in coordination of copper. Direct involvement of the N-terminal domains in the binding of copper suggests their important role in copper-dependent functions of human copper-transporting adenosine triphosphatases (Wilson's and Menkes disease proteins).

Highlights

N-terminal domains of the Wilson’s and Menkes disease proteins (N-WND and N-MNK) were overexpressed in a soluble form in Escherichia coli as fusions with maltose-binding protein, purified, and their metal-binding properties were characterized
Expression of the N-terminal Domains of Human copperATPases in Soluble Form—Our initial attempt to express NWND and N-MNK proteins in E. coli led to the accumulation of protein in inclusion bodies under all experimental conditions we tested
We were successful in using this system for both N-WND and N-MNK metalbinding domains, and after induction with isopropyl-␤-D-thiogalactopyranoside, we observed a significant level of expression of both fusion proteins (Fig. 2A)

Summary

EXPERIMENTAL PROCEDURES

Expression of Heavy-metal Binding Domains of the WND and MNK Proteins—To create fusion proteins of the N-WND and N-MNK with the maltose-binding protein (MBP), segments corresponding to 64 –1868 base pairs of the WND cDNA and 1–1836 base pairs of the MNK cDNA were amplified by polymerase chain reaction and incorporated into pMAL-c2 vector (New England Biolabs) at the 3Ј-end of coding sequence for the maltose-binding protein (MBP), after the factor Xa cleavage site. Involvement of Cysteine Residues—The involvement of cysteine residues in coordination of copper was demonstrated by the ability of different heavy metals to protect cysteine residues in the metal-binding domains against labeling with the cysteine-directed fluorescent reagent 7-diethylamino-3- (4Ј-maleimidylphenyl)-4-methylcoumarin (CPM; Molecular Probes) For these experiments, purified N-WND and N-MNK proteins were incubated in the presence of increasing concentrations of different heavy metals (see legend for Fig. 7.) and pulse-labeled with a 10 –20 molar excess of CPM with respect to protein for 1 min in the dark. A difference in the absorbance of copper-BCA complex at 562 nm in the presence and absence of ascorbate points to the Cu(II) form of released cation This procedure has been used to measure the in vivo and in vitro binding of copper to the domains. After 10 –15 min incubation at room temperature, unbound copper was removed by overnight dialysis at 4 °C, and bound copper was measured with BCA in the presence of 1 mM ascorbate

RESULTS

DISCUSSION

Kaplan