Distinct phenotype of a Wilson disease mutation reveals a novel trafficking determinant in the copper transporter ATP7B

Abstract

Wilson disease (WD) is a monogenic autosomal-recessive disorder of copper accumulation that leads to liver failure and/or neurological deficits. WD is caused by mutations in ATP7B, a transporter that loads Cu(I) onto newly synthesized cupro-enzymes in the trans-Golgi network (TGN) and exports excess copper out of cells by trafficking from the TGN to the plasma membrane. To date, most WD mutations have been shown to disrupt ATP7B activity and/or stability. Using a multidisciplinary approach, including clinical analysis of patients, cell-based assays, and computational studies, we characterized a patient mutation, ATP7B(S653Y), which is stable, does not disrupt Cu(I) transport, yet renders the protein unable to exit the TGN. Bulky or charged substitutions at position 653 mimic the phenotype of the patient mutation. Molecular modeling and dynamic simulation suggest that the S653Y mutation induces local distortions within the transmembrane (TM) domain 1 and alter TM1 interaction with TM2. S653Y abolishes the trafficking-stimulating effects of a secondary mutation in the N-terminal apical targeting domain. This result indicates a role for TM1/TM2 in regulating conformations of cytosolic domains involved in ATP7B trafficking. Taken together, our experiments revealed an unexpected role for TM1/TM2 in copper-regulated trafficking of ATP7B and defined a unique class of WD mutants that are transport-competent but trafficking-defective. Understanding the precise consequences of WD-causing mutations will facilitate the development of advanced mutation-specific therapies.

Keywords: ceruloplasmin; interdomain interactions; molecular dynamics.

Fig. 1.

Hypothetical ATP7B model and multiple species alignment of the conserved regions, amino acids 621–668, in the two Cu-ATPases. (A) A hypothetical ATP7B ribbon model, generated by UCSF Chimera, showing the conserved core organization (20). The two large cytoplasmic loops in the core structure are: the A domain (actuator, green), between TM4 and TM5, which contains the phosphatase activity; and the N and P domains (nucleotide binding and phosphorylation, red) between TM6 and TM7, which bind ATP (N), catalyzing formation of a phosphorylated intermediate (P) as part of the catalytic cycle. The eight TMs (yellow are): TM1 (amino acids 645–670), TM2 (including the platform helix, amino acids 694–722), TM3 (amino acids 729–749), TM4 (amino acids 765–786), TM5 (amino acids 916–942), TM6 (amino acids 967–1004), TM7 (amino acids 1307–1345), and TM8 (amino acids 1352–1373). The six N-terminal MBDs (blue, N-MBDs, also referred to as the N-terminal domain of ATP7B, N-ATP7B) (61, 62) were manually positioned onto the published model. The box approximates the region of the multiple species alignment shown in B. (B) A multiple species alignment of human ATP7B sequence 621–668 (Upper) and ATP7A sequence 621–668 (Lower). WD patient mutations are underlined and in bold. The ATP7B S653 position is marked with an asterisk (bold). Portions of MBD6 and TMD 1 are bracketed. In ATP7A, the bracketed sequence shows the deleted region that is replaced with two amino acids (IR) in a patient with Occipital Horn Syndrome. The deleted sequence of ATP7A is underlined and in bold (35). Alignments were obtained using ClustalW (63). Amino acids that are identical (*), conserved (:), and semiconserved (.) are shown.

Fig. 2.

wtATP7B and ATP7B^S653Y have similar Cu(I) transport activity yet show different trafficking behaviors in polarized hepatic cells. (C) ATP7B^S653Y exhibits Cu(I) transport activity. YST cells were cotransfected with pTyrosinase (pTyr) and the indicated plasmid as described in Methods. The black reaction product reflects copper-dependent tyrosinase activity. Negative controls (A and D) and wtATP7B (B) were always included. (Magnification: A–D, 40×.) WIF-B cells were infected with wtATP7B and ATP7B^S653Y adenovirus, cultured overnight in 10 μM BCS, then kept in the chelator (E, E′, G, and G′), or incubated in 10 μM CuCl₂ (F and F′) or 100 μM CuCl₂ (H and H′) for 1 h, fixed, stained with antibodies to GFP (green in E, F, G, and H) and TGN38 (red in E′, F′, G′, and H′), and imaged by confocal microscopy. Single optical sections are shown. Exogenous wtATP7B redistributes from the TGN (E and E′) to vesicles and the apical membrane when copper levels are elevated (F and F′). In contrast, ATP7B^S653Y remains in the TGN in the absence (G and G′) or presence of copper (H and H′). n, nucleus; *, apical space. (Scale bars, 10 μm.)

Fig. 3.

Qualitative and quantitative evidence that five WD patient mutations show normal trafficking but ATP7B^S653Y does not. (A and B) WIF-B cells infected with adenoviruses encoding wtATP7B and each of the patient mutations were processed for anterograde and retrograde trafficking as described in Methods. (A and A′) In the presence of copper, GFP-ATP7B^G626A fluorescence is seen at the apical membrane and in vesicles; it does not overlap with the TGN marker (red). (B and B′) After copper chelation, the protein is no longer seen at the apical membrane and overlaps strongly with the TGN marker. (C) The numbers of polar cells with ATP7B protein fluorescence at the apical surface in the presence and after copper chelation were determined and expressed as a percentage of the total polarized cells expressing the ATP7B protein. Five of the mutant ATP7B showed wild-type trafficking in copper and after copper chelation. However, the ATP7B^S653Y mutant was not found at the apical surface under any copper condition; it was not evaluated using the retrograde assay. The numbers of polar cells evaluated for anterograde/retrograde trafficking, respectively, were: G626A: 310/152; H639Y: 193/172; L641: 202/118; D642H: 242/168; M645R: 228/123; and S653Y: 228. (Scale bar, 10 μm.)

Fig. 4.

The ATP7B structural model shows that Ser653 in TM1 interacts with different TM2 residues than does the patient mutation, Tyr653. (A) A rotated view of the ATP7B ribbon model shows the full atom sphere of S653 (magenta) and the platform helix (yellow). (B and C) Expanded views show interatomic distances among selected residues nearby Ser653 (B) and Tyr653 (C). (B) In Ser653, there is a 3 Å distance between the hydroxyl group γ-oxygen (red) of Ser653 (magenta) and the carbonyl oxygen (red) of Gly710 (magenta) just before the platform helix (yellow). Tyr713 (magenta) is part of the platform helix (yellow). (C) However, Tyr653 (blue) is rotated away from Gly710 (magenta) and shows a large interatomic distance of 6.8 Å with it. Instead, Tyr653 interacts with Tyr713 (magenta) as revealed by an interatomic distance of 3.7 Å between their functional groups (red). All models were generated using PyMOL (The PyMOL Molecular Graphics System, v1.5.0.4; Schrödinger).

Fig. 5.

ATP7B^S653F and ATP7B^S653E mutant proteins are retained in the TGN in 100-μM copper, whereas ATP7B^S653A mutant protein exhibits wild-type trafficking. WIF-B cells were infected with the indicated ATP7B adenovirus, cultured, and processed as described in Methods. Single confocal sections are shown. (A) Substitution of S653 with either phenylalanine (F, Phe) or glutamate (E, Glu) blocked TGN-exit of ATP7B in 100 μM copper. A cytoplasmic green haze found in ATP7B^S653E-expressing cells may reflect its degradation (Fig. S3A). (B) In contrast, ATP7B^S653A redistributed from the TGN (B, Left) to vesicles and the apical membrane when copper levels were elevated (B, Right). n, nucleus; *, apical space. (Scale bar,10 μm.) (C) Quantitative evidence that ATP7B^S653A, ATP7B^S653T and ATP7B^S653C substitutions show wild-type trafficking in polar hepatic cells. WIF-B cells infected with adenoviruses encoding wtATP7B and three 653 substitutions were cultured, processed, and quantitatively analyzed for trafficking as described in Fig. 3. Like wtATP7B, ATP7B^S653A, ATP7B^S653T, and ATP7B^S653C variants exited the TGN (anterograde) in 10-μM copper and returned (retrograde) when copper levels were lowered. The number of polar cells evaluated for anterograde/retrograde trafficking, respectively, were Ala: 108/115; Thr: 98/142; and Cys: 158/77.

Fig. 6.

Molecular dynamic simulation of Ser653 mutant proteins reveals fluctuations in the SASA. (A) Box plots show fluctuations of SASAs at the 653 position in wild-type and mutant (Ala, Cys, Thr, Tyr, Phe, Glu, and Asp) substituted models. Tyr and Phe mutants are more exposed and Glu is buried within the structure. (B) Box plots showing fluctuations of SASAs at the 713 tyrosine in wild-type and mutant (Ala, Cys, Thr, Tyr, Phe, Glu, and Asp) substituted models. Solid line represents median; dashed line with dot represents average; upper dot represents value at 95%; lower dot represents value at 5%; error bars are shown. The 95th and 5th percentile datapoints represent the sampling extremes.

Fig. 7.

ATP7B^S653Y exhibits a dominant effect upon the trafficking of ATP7B^Y44A but not ATP7B^TGE>AAA. WIF-B cells were infected with ATP7B^TGE>AAA (A, A′, B, and B′), ATP7B^{S653Y/TGE>AAA} (C, C′, D, and D′), ATP7B^Y44A (E, E′, F, and F′), and ATP7B^S653Y/Y44A (G, G′, H, and H′) adenoviruses, cultured overnight in 10 μM BCS, then either maintained in BCS (A, A′, C, C′, E, E′, G, and G′) or incubated in 10 μM CuCl₂ (B, B′, D, D′, F, F′, H, and H′) for 1 h before being fixed and processed as described in Fig. 2. (A and B) ATP7B^TGE>AAA trafficked to vesicles and the apical membrane in the absence (A and A′) or presence of copper (B, B′). Quantification showed that in 85% (n = 118) and 91% (n = 107) of polar cells expressing ATP7B^TGE>AAA irrespective of copper’s presence or absence, the protein was in vesicles and the apical surface. (C and D) The double-mutant, ATP7B^{S653Y/TGE>AAA}, was also found in vesicles and the apical surface in the absence (C and C′) or presence of copper (D and D′). In the absence but not the presence of copper (C and C′), ATP7B^{S653Y/TGE>AAA} was also found in the ER in 55% of expressing, polar cells (n = 62). (E and F) ATP7B^Y44A trafficked to the basolateral PM (76% of cells n = 59) in the absence (E and E′) or presence of copper (F and F′). (G and H) Introduction of S653Y in ATP7B^Y44A changed the resulting double-mutant protein’s behavior. ATP7B^S653Y/Y44A was found predominantly in the TGN in the absence of copper (G and G′) and in its presence (H and H′). Only 22% (n = 60) of polar cells expressing ATP7B^S653Y/Y44A showed basolateral PM fluorescence. n, nucleus; *, apical space. (Scale bar, 10 μm.)