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Kinetic analysis of the interaction of the copper chaperone Atox1 with the metal binding sites of the Menkes protein

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journal contribution
posted on 2003-06-06, 00:00 authored by D Strausak, M Howie, Stephen Firth, A Schlicksupp, R Pipkorn, G Multhaup, Julian MercerJulian Mercer
Excess copper is effluxed from mammalian cells by the Menkes or Wilson P-type ATPases (MNK and WND, respectively). MNK and WND have six metal binding sites (MBSs) containing a CXXC motif within their N-terminal cytoplasmic region. Evidence suggests that copper is delivered to the ATPases by Atox1, one of three cytoplasmic copper chaperones. Attempts to monitor a direct Atox1-MNK interaction and to determine kinetic parameters have not been successful. Here we investigated interactions of Atox1 with wild-type and mutated pairs of the MBSs of MNK using two different methods: yeast two-hybrid analysis and real-time surface plasmon resonance (SPR). A copper-dependent interaction of Atox1 with the MBSs of MNK was observed by both approaches. Cys to Ser mutations of conserved CXXC motifs affected the binding of Atox1 underlining the essentiality of Cys residues for the copper-induced interaction. Although the yeast two-hybrid assay failed to show an interaction of Atox1 with MBS5/6, SPR analysis clearly demonstrated a copper-dependent binding with all six MBSs highlighting the power and sensitivity of SPR as compared with other, more indirect methods like the yeast two-hybrid system. Binding constants for copper-dependent chaperone-MBS interactions were determined to be 10–5-10–6 M for all the MBSs representing relatively low affinity binding events. The interaction of Atox1 with pairs of the MBSs was non-cooperative. Therefore, a functional difference of the MBSs in the MNK N terminus cannot be attributed to cooperativity effects or varying affinities of the copper chaperone Atox1 with the MBSs.

History

Journal

Journal of biological chemistry

Volume

278

Pagination

20821 - 20827

Location

Baltimore, Md.

Open access

  • Yes

ISSN

0021-9258

eISSN

1083-351X

Language

eng

Publication classification

C1 Refereed article in a scholarly journal

Copyright notice

2003 by the American Society for Biochemistry and Molecular Biology