TY - JOUR
T1 - Copper transfer to the N-terminal domain of the Wilson disease protein (ATP7B)
T2 - X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands
AU - Ralle, Martina
AU - Lutsenko, Svetlana
AU - Blackburn, Ninian J.
N1 - Funding Information:
The work was supported by a Program Project Grant P01-GM067166 to N.J.B. and S.L. We thank Deepali Datta for assistance in preparation of HAH1. We thank Dr James E. Penner-Hahn for making available the XAS data on the bis-tetramethylbenzenethiolate–Cu(I) model complex, and Dr Graham George for making available the XAS data on the tris-tetramethylthiourea–Cu(I) complex. We gratefully acknowledge the use of facilities at the Stanford Synchrotron Radiation Laboratory, which is supported by the National Institutes of Health Biomedical Research Technology Program, Division of Research Resources, and by the US Department of Energy, Basic Energy Sciences (BES) and Office of Biological and Environmental Research (OBER).
PY - 2004/5
Y1 - 2004/5
N2 - The copper-transporting ATPases are 165-175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu-Cu interaction which may signal a conformational change of the N-terminal domain.
AB - The copper-transporting ATPases are 165-175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu-Cu interaction which may signal a conformational change of the N-terminal domain.
KW - Copper Chaperone ATPase EXAFS Wilson
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U2 - 10.1016/j.jinorgbio.2004.02.009
DO - 10.1016/j.jinorgbio.2004.02.009
M3 - Article
C2 - 15134922
AN - SCOPUS:2342549621
SN - 0162-0134
VL - 98
SP - 765
EP - 774
JO - Journal of Inorganic Biochemistry
JF - Journal of Inorganic Biochemistry
IS - 5
ER -