TY - JOUR
T1 - Human copper transporter ATP7B (Wilson disease protein) forms stable dimers in vitro and in cells
AU - Jayakanthan, Samuel
AU - Braiterman, Lelita T.
AU - Hasan, Nesrin M.
AU - Unger, Vinzenz M.
AU - Lutsenko, Svetlana
N1 - Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/11/17
Y1 - 2017/11/17
N2 - ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single- chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1-4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1-4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2. Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu- ATPase, positions of the main domains, and a dimer interface.
AB - ATP7B is a copper-transporting P1B-type ATPase (Cu-ATPase) with an essential role in human physiology. Mutations in ATP7B cause the potentially fatal Wilson disease, and changes in ATP7B expression are observed in several cancers. Despite its physiologic importance, the biochemical information about ATP7B remains limited because of a complex multidomain organization of the protein. By analogy with the better characterized prokaryotic Cu-ATPases, ATP7B is assumed to be a single- chain monomer. We show that in eukaryotic cells, human ATP7B forms dimers that can be purified following solubilization. Deletion of the four N-terminal metal-binding domains, characteristic for human ATP7B, does not disrupt dimerization, i.e. the dimer interface is formed by the domains that are conserved among Cu-ATPases. Unlike the full-length ATP7B, which is targeted to the trans-Golgi network, 1-4ΔMBD-7B is targeted primarily to vesicles. This result and the analysis of differentially tagged ATP7B variants indicate that the dimeric structure is retained during ATP7B trafficking between the intracellular compartments. Purified dimeric species of 1-4ΔMBD-7B were characterized by a negative stain electron microscopy in the presence of ADP/MgCl2. Single-particle analysis yielded a low-resolution 3D model that provides the first insight into an overall architecture of a human Cu- ATPase, positions of the main domains, and a dimer interface.
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U2 - 10.1074/jbc.M117.807263
DO - 10.1074/jbc.M117.807263
M3 - Article
C2 - 28842499
AN - SCOPUS:85034435614
SN - 0021-9258
VL - 292
SP - 18760
EP - 18774
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 46
ER -