title: Difference in stability of the N-domain underlies distinct intracellular properties of the E1064A and H1069Q mutants of copper-transporting ATPase ATP7B.
abstract: Wilson disease (WD) is a disorder of copper metabolism caused by mutations in the Cu-transporting ATPase ATP7B. WD is characterized by significant phenotypic variability, the molecular basis of which is poorly understood. The E1064A mutation in the N-domain of ATP7B was previously shown to disrupt ATP binding. We have now determined, by NMR, the structure of the N-domain containing this mutation and compared properties of E1064A and H1069Q, another mutant with impaired ATP binding. The E1064A mutation does not change the overall fold of the N-domain. However, the position of the α1,α2-helical hairpin (α-HH) that houses Glu(1064) and His(1069) is altered. The α-HH movement produces a more open structure compared with the wild-type ATP-bound form and misaligns ATP coordinating residues, thus explaining complete loss of ATP binding. In the cell, neither the stability nor targeting of ATP7B-E1064A to the trans-Golgi network differs significantly from the wild type. This is in a contrast to the H1069Q mutation within the same α-HH, which greatly destabilizes protein both in vitro and in cells. The difference between two mutants can be linked to a lower stability of the α-HH in the H1069Q variant at the physiological temperature. We conclude that the structural stability of the N-domain rather than the loss of ATP binding plays a defining role in the ability of ATP7B to reach the trans-Golgi network, thus contributing to phenotypic variability in WD.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Saskatchewan, Canada. oleg.dmitriev@usask.ca
authors.forename: Oleg Y
authors.initials: OY
authors.lastname: Dmitriev
authors.forename: Ashima
authors.initials: A
authors.lastname: Bhattacharjee
authors.forename: Sergiy
authors.initials: S
authors.lastname: Nokhrin
authors.forename: Eva-Maria E
authors.initials: EM
authors.lastname: Uhlemann
authors.forename: Svetlana
authors.initials: S
authors.lastname: Lutsenko
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000073840:Copper-Transporting ATPases; D057809:HEK293 Cells; D006527:Hepatolenticular Degeneration / Q000201:enzymology / Q000235:genetics; D006801:Humans; D020125:Mutation, Missense; D011485:Protein Binding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D021601:trans-Golgi Network / Q000201:enzymology / Q000235:genetics
chemical_list: D027682:Cation Transport Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C000612464:ATP7B protein, human; D000073840:Copper-Transporting ATPases