Abstract
The copper-transporting ATPase ATP7B is essential for normal distribution of copper in human cells. Mutations in ATP7B lead to Wilson's disease, a severe disorder with neurological and hepatic manifestations. One of the most common disease mutations, a H1069Q substitution, causes intracellular mislocalization of ATP7B (the Wilson's disease protein, WNDP). His-1069 is located in the nucleotide-binding domain of WNDP and is conserved in all copper-transporting ATPases from bacteria to mammals; however, the specific role of this His in the structure and function of WNDP remains unclear. We demonstrate that substitution of His-1069 for Gln, Ala, or Cys does not significantly alter the folding of the WNDP nucleotide-binding domain or the proteolytic resistance of the full-length WNDP. In contrast, the function of WNDP is markedly affected by the mutations. The ability to form an acylphosphate intermediate in the presence of ATP is entirely lost in all three mutants, suggesting that His-1069 is important for ATP-dependent phosphorylation. Other steps of the WNDP enzymatic cycle are less dependent on His-1069. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, beta,gamma-imidoadenosine 5'-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP. Although binding of AMP-PNP is not disrupted by the mutation, the apparent affinity for the nucleotide is decreased by 4-fold. We conclude that His-1069 is responsible for proper orientation of ATP in the catalytic site of WNDP prior to ATP hydrolysis.
Highlights
Copper is a cofactor of important metabolic enzymes that are involved in a variety of physiological processes such as radical detoxification, oxidative phosphorylation, and iron metabolism
The Effect of the His-1069 Substitution on Folding of the ATP-binding Domain—As shown in Fig. 1, His-1069 is located in the ATP-binding domain of WNDP (ATP-BD) and could be essential for correct folding of this important functional domain
Analysis of protein folding using circular dichroism spectroscopy (CD) spectroscopy revealed that overall secondary structure of all mutants is similar to the wt ATP-BD structure (Fig. 2A, and Table I), indicating that the mutations do not cause marked misfolding of this domain
Summary
Copper is a cofactor of important metabolic enzymes that are involved in a variety of physiological processes such as radical detoxification, oxidative phosphorylation, and iron metabolism. The H1069C mutant shows normal phosphorylation in the presence of inorganic phosphate; it binds an ATP analogue, ,␥imidoadenosine 5-triphosphate (AMP-PNP), and copper and undergoes nucleotide-dependent conformational transitions similar to those of the wild-type WNDP.
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