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

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

Research output: Contribution to journalArticlepeer-review

210 Scopus citations

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).

Original languageEnglish (US)
Pages (from-to)18939-18944
Number of pages6
JournalJournal of Biological Chemistry
Volume272
Issue number30
DOIs
StatePublished - 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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