A Luminal Loop of Wilson Disease Protein Binds Copper and Is Required for Protein Activity.

Abstract	The copper-transporting ATPase ATP7B is essential for loading of copper ions to copper-dependent enzymes in the secretory pathway; its inactivation results in Wilson disease. In contrast to copper-ion uptake by the cytoplasmic domains, ATP7B-mediated copper-ion release in the Golgi has not been explored yet. We demonstrate here that a luminal loop in ATP7B, rich in histidine/methionine residues, binds reduced copper (Cu(I)) ions, and identified copper-binding residues play an essential role in ATP7B-mediated metal ion release. NMR experiments on short-peptide models demonstrate that three methionine and two histidine residues are specifically involved in Cu(I) ion binding; with these residues replaced by alanines, no Cu(I) ion interaction is detected. Although more than one Cu(I) ion can interact with the wild-type peptide, removing either all histidine or all methionine residues reduces the stoichiometry to one Cu(I) ion binding per peptide. Using a yeast complementation assay, we show that for efficient copper transport by full-length ATP7B, the complete set of histidine and methionine residues in the lumen loop are required. The replacement of histidine/methionine residues by alanines does not perturb overall ATP7B structure, as the localization of ATP7B variants in yeast cells matches that of the wild-type protein. Thus, in similarity to ATP7A, ATP7B also appears to have a luminal "exit" copper ion site.
Authors	Birgit Köhn, Kumaravel Ponnandai Shanmugavel, Min Wu, Michael Kovermann, Pernilla Wittung-Stafshede
Journal	Biophysical journal (Biophys J) Vol. 115 Issue 6 Pg. 1007-1018 (09 18 2018) ISSN: 1542-0086 [Electronic] United States
PMID	30173886 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Copyright	Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Chemical References	Copper Copper-Transporting ATPases
Topics	Amino Acid Sequence Binding Sites Copper (metabolism) Copper-Transporting ATPases (chemistry, metabolism) Humans Models, Molecular Protein Binding Protein Conformation, alpha-Helical Substrate Specificity

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