A luminal loop of Wilson disease protein binds copper and is required for protein activity

October 11, 2018


A luminal loop of Wilson disease protein binds copper and is required for protein activity


Birgit Köhn, Kumaravel Ponnandai Shanmugavel, Min Wu, Michael Kovermann, Pernilla Wittung-Stafshede




Biophysical Journal


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 here demonstrate 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, also ATP7B appears to have a luminal ‘exit’ copper ion site.




Circular dichroism, Secondary structure, Biochemistry