title: The distinct functional properties of the nucleotide-binding domain of ATP7B, the human copper-transporting ATPase: analysis of the Wilson disease mutations E1064A, H1069Q, R1151H, and C1104F.
abstract: Copper transport by the P(1)-ATPase ATP7B, or Wilson disease protein (WNDP),1 is essential for human metabolism. Perturbation of WNDP function causes intracellular copper accumulation and severe pathology, known as Wilson disease (WD). Several WD mutations are clustered within the WNDP nucleotide-binding domain (N-domain), where they are predicted to disrupt ATP binding. The mechanism by which the N-domain coordinates ATP is presently unknown, because residues important for nucleotide binding in the better characterized P(2)-ATPases are not conserved within the P(1)-ATPase subfamily. To gain insight into nucleotide binding under normal and disease conditions, we generated the recombinant WNDP N-domain and several WD mutants. Using isothermal titration calorimetry, we demonstrate that the N-domain binds ATP in a Mg(2+)-independent manner with a relatively high affinity of 75 microm, compared with millimolar affinities observed for the P(2)-ATPase N-domains. The WNDP N-domain shows minimal discrimination between ATP, ADP, and AMP, yet discriminates well between ATP and GTP. Similar results were obtained for the N-domain of ATP7A, another P(1)-ATPase. Mutations of the invariant WNDP residues E1064A and H1069Q drastically reduce nucleotide affinities, pointing to the likely role of these residues in nucleotide coordination. In contrast, the R1151H mutant exhibits only a 1.3-fold reduction in affinity for ATP. The C1104F mutation significantly alters protein folding, whereas C1104A does not affect the structure or function of the N-domain. Together, the results directly demonstrate the phenotypic diversity of WD mutations within the N-domain and indicate that the nucleotide-binding properties of the P(1)-ATPases are distinct from those of the P(2)-ATPases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, OR 97239-3098, USA.
authors.forename: Clinton T
authors.initials: CT
authors.lastname: Morgan
authors.forename: Ruslan
authors.initials: R
authors.lastname: Tsivkovskii
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Kosinsky
authors.forename: Roman G
authors.initials: RG
authors.lastname: Efremov
authors.forename: Svetlana
authors.initials: S
authors.lastname: Lutsenko
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D002151:Calorimetry; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics*; D002942:Circular Dichroism; D000073840:Copper-Transporting ATPases; D003545:Cysteine / Q000737:chemistry; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature
chemical_list: D027682:Cation Transport Proteins; D009711:Nucleotides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D000255:Adenosine Triphosphate; D001120:Arginine; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases; D008274:Magnesium; D003545:Cysteine