High Yield Heterologous Expression of Wild-type and Mutant Cu+-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation

ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7...

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Bibliographic Details
Published in:The Journal of biological chemistry 2009-08, Vol.284 (32), p.21307-21316
Main Authors: Pilankatta, Rajendra, Lewis, David, Adams, Christopher M., Inesi, Giuseppe
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - biosynthesis
Amino Acid Sequence
Animals
Cation Transport Proteins - biosynthesis
Chlorocebus aethiops
Copper - chemistry
Copper-Transporting ATPases
COS Cells
Gene Expression Regulation, Enzymologic
Humans
Hydrogen-Ion Concentration
Membrane Transport, Structure, Function, and Biogenesis
Microsomes - metabolism
Molecular Sequence Data
Mutation
Phosphorylation
Protein Conformation
Protein Structure, Tertiary
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Summary:ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10–20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of Pi/mg of protein/min at 37 °C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [32P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser478 and Ser481 (NMBD), Ser1121 (“N” domain) and Ser1453 (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M109.023341