title: The Lys1010-Lys1325 fragment of the Wilson's disease protein binds nucleotides and interacts with the N-terminal domain of this protein in a copper-dependent manner.
abstract: Wilson's disease, an autosomal disorder associated with vast accumulation of copper in tissues, is caused by mutations in a gene encoding a copper-transporting ATPase (Wilson's disease protein, WNDP). Numerous mutations have been identified throughout the WNDP sequence, particularly in the Lys(1010)-Lys(1325) segment; however, the biochemical properties and molecular mechanism of WNDP remain poorly characterized. Here, the Lys(1010)-Lys(1325) fragment of WNDP was overexpressed, purified, and shown to form an independently folded ATP-binding domain (ATP-BD). ATP-BD binds the fluorescent ATP analogue trinitrophenyl-ATP with high affinity, and ATP competes with trinitrophenyl-ATP for the binding site; ADP and AMP appear to bind to ATP-BD at the site separate from ATP. Purified ATP-BD hydrolyzes ATP and interacts specifically with the N-terminal copper-binding domain of WNDP (N-WNDP). Strikingly, copper binding to N-WNDP diminishes these interactions, suggesting that the copper-dependent change in domain-domain contact may represent the mechanism of WNDP regulation. In agreement with this hypothesis, N-WNDP induces conformational changes in ATP-BD as evidenced by the altered nucleotide binding properties of ATP-BD in the presence of N-WNDP. Significantly, the effects of copper-free and copper-bound N-WNDP on ATP-BD are not identical. The implications of these results for the WNDP function are discussed.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
authors.forename: R
authors.initials: R
authors.lastname: Tsivkovskii
authors.forename: B C
authors.initials: BC
authors.lastname: MacArthur
authors.forename: S
authors.initials: S
authors.lastname: Lutsenko
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D027682:Cation Transport Proteins; D003300:Copper / Q000378:metabolism*; D000073840:Copper-Transporting ATPases; D017931:DNA Primers; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D002352:Carrier Proteins; D027682:Cation Transport Proteins; D017931:DNA Primers; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D003300:Copper; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000073840:Copper-Transporting ATPases; D008239:Lysine