Expression, Purification, and Metal Binding Properties of the N-terminal Domain from the Wilson Disease Putative Copper-transporting ATPase (ATP7B)

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast twohybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.

Section: Discussionmentioning

confidence: 99%

Copper-dependent Interaction of Dynactin Subunit p62 with the N Terminus of ATP7B but Not ATP7A

Lim

Cater

Mercer

et al. 2006

“…A similar function has been proposed for the cytosolic amino-terminal metal binding domains in the coppertransporting P-type ATPases Ccc2/ATP7A/B, which are multimembrane-spanning proteins localized in the trans-Golgi network and responsible for copper translocation into the lumen of the secretory pathway (42). Human ATP7A, or MNK protein, which is defective in patients with Menkes syndrome, contains six metal binding domains that bind Cu(I) (43)(44)(45)(46) and are absolutely required under the low bioavailable copper concentrations of the cytosol, but apparently dispensable for the in vitro catalytic activity of the protein (47). Copper binding studies and structural data will be important to test a copper binding model for the Ctr1 Mets motif function.…”

Section: Function Of Conserved Methionine Residues In Humanmentioning

confidence: 99%

Biochemical and Genetic Analyses of Yeast and Human High Affinity Copper Transporters Suggest a Conserved Mechanism for Copper Uptake

Puig¹,

Lee²,

Lau

et al. 2002

351

459

The redox active metal copper is an essential cofactor in critical biological processes such as respiration, iron transport, oxidative stress protection, hormone production, and pigmentation. A widely conserved family of high affinity copper transport proteins (Ctr proteins) mediates copper uptake at the plasma membrane. However, little is known about Ctr protein topology, structure, and the mechanisms by which this class of transporters mediates high affinity copper uptake. In this report, we elucidate the topological orientation of the yeast Ctr1 copper transport protein. We show that a series of clustered methionine residues in the hydrophilic extracellular domain and an MXXXM motif in the second transmembrane domain are important for copper uptake but not for protein sorting and delivery to the cell surface. The conversion of these methionine residues to cysteine, by site-directed mutagenesis, strongly suggests that they coordinate to copper during the process of metal transport. Genetic evidence supports an essential role for cooperativity between monomers for the formation of an active Ctr transport complex. Together, these results support a fundamentally conserved mechanism for high affinity copper uptake through the Ctr proteins in yeast and humans.

“…Domains 1-4 of WND have also been proposed to function in copper-responsive localization (27,28). Although all six metal-binding domains can bind copper (22,34), their copper-binding affinities are not known. Furthermore, the copper-binding affinity of Atox1 has not been reported.…”

mentioning

confidence: 99%

Binding of Copper(I) by the Wilson Disease Protein and Its Copper Chaperone

Wernimont¹,

Yatsunyk

Rosenzweig

2004

The Wilson disease protein (WND) is a transport ATPase involved in copper delivery to the secretory pathway. Mutations in WND and its homolog, the Menkes protein, lead to genetic disorders of copper metabolism. The WND and Menkes proteins are distinguished from other P-type ATPases by the presence of six soluble N-terminal metal-binding domains containing a conserved CXXC metal-binding motif. The exact roles of these domains are not well established, but possible functions include exchanging copper with the metallochaperone Atox1 and mediating copper-responsive cellular relocalization. Although all six domains can bind copper, genetic and biochemical studies indicate that the domains are not functionally equivalent. One way the domains could be tuned to perform different functions is by having different affinities for Cu(I). We have used isothermal titration calorimetry to measure the association constant (K a ) and stoichiometry (n) values of Cu(I) binding to the WND metal-binding domains and to their metallochaperone Atox1. The association constants for both the chaperone and target domains are ϳ10 5 to 10 6 M ؊1 , suggesting that the handling of copper by Atox1 and copper transfer between Atox1 and WND are under kinetic rather than thermodynamic control. Although some differences in both n and K a values are observed for variant proteins containing less than the full complement of six metal-binding domains, the data for domains 1-6 were best fitted with a single site model. Thus, the individual functions of the six WND metal-binding domains are not conferred by different Cu(I) affinities but instead by fold and electrostatic surface properties.