Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications
Introduction
Wilson disease is a genetic disorder of copper metabolism characterized by the toxic accumulation of copper in the liver and then in extrahepatic sites. The gene responsible for Wilson disease, located on chromosome 13, was identified in 1993 [1], [2], [3] and found to encode a copper transporting P-type ATPase (ATP7B). ATP7B is localized in the trans-Golgi network of hepatocytes under low copper conditions, redistributes to cytoplasmic vesicles when cells are exposed to elevated copper levels, and then recycles back to trans-Golgi network when copper is removed [4], [5], [6].
The predicted structure of the Wilson disease copper-transporting ATPase contains general features conserved among members of the P-type ATPase family. These are the TGEA motif (actuator domain), the DKTGT and TGDN motifs (phosphorylation domain) and the sequence MXGDGXNDXP found in the hinge region that connects the phosphorylation domain to the transmembrane segment. ATP7B is further classified as a heavy metal transporting P-type ATPase since it contains six metal-binding motifs, GMTCXXC, at the N-terminus of the molecule, the CPC motif in the sixth intramembrane region (TM6), the SEHPL motif in the nucleotide binding domain and eight transmembrane segments [4], [5] (Fig. 1). Studies of site-directed mutagenesis have shown that the repeats closest to the transmembrane domain, the WD6 in particular, appear to be the most important for copper transport across the membrane [7].
One of the earliest structures to be made available for metal binding proteins was that of the mercury binding protein MerP [8]. The 3D structure of the 18-residue linear peptide used as a model of the metal-binding loop of MerP, has shown that when Hg(II) binds the sequence CXXC, imposes a highly stable unique conformation to the latter similar to the 72-residue protein [9]. The peptides' ability to mimic the metal-binding loops highlights their utility as important tools to study the interactions of protein metal-binding sites with Cu(I). In a similar manner the 2K10p peptide has been synthesized, containing the residues GMTCASCVHN from the WD6 of ATP7B in order to study the interaction of metal-binding sites with Cu(I) (Fig. 1).
The construction of chimeric proteins consisting of the ZntA (zinc transporting ATPase) core domain and the amino-terminal domain of ATP7B resulted in the maintenance of the ability to transport zinc, but not copper [10], thus illustrating that the transmembrane segment is the determinant of the metal specificity observed for P-type ATPases. The fourth transmembrane domain of the Ca-ATPase [11], Na,K-ATPase [12] and H-ATPase [13] is predicted to correspond to transmembrane domain six of ATP7B, and contain a conserved proline residue found in all P-type ATPases [14]. This transmembrane domain is associated with the transduction channel and contains residues critical to cation binding. In heavy metal-transporting ATPases a pair of cysteine residues flanks the conserved proline to form the highly conserved CPC motif. Mutations of the CPC residues result in non-functional proteins [15], [16], [17] that are unable to redistribute in response to copper [18], [19]. Mammalian copper-transporting ATPases have an additional conserved cysteine, forming a CXXCPC motif.
In order to investigate the role of each of the 3 cysteines present in the TM6 of ATP7B, the 2K8p peptide (Fig. 2), and three mutants of 2K8p (Fig. 2) were synthesized in which one cysteine residue at a time is replaced by a serine residue. Due to the importance of the metal-binding sites in the function of Wilson ATPase, the investigation of the nature of their interaction with Cu(I) comprises an essential step in the elucidation of the mechanism of regulation of this protein and it's mechanism of action. Following this concept, the comparison of the interactions of all five of the above mentioned peptides (Fig. 2) with Cu(I) has been the focus of this study.
Section snippets
Homology modeling
The 2K10p and 2K8p peptides were modeled by homology using SWISS-Model and SWISS-PDB-viewer [20], [21], [22] to generate pictorial representations of the peptides as previously described by Fatemi and Sarkar [23].
Preparation of peptides
In all peptide sequences two lysine residues were added at both termini to increase the solubility in aqueous media. All peptides were prepared by solid phase peptide synthesis utilizing a peptide synthesizer (Pioneer) according to the Fmoc-strategy. Fmoc-PAL-PEG-PS resin was used as
Homology modeling
2K10p is shown in Fig. 3(a) as it is found in a homology model of WD6, the sixth Cu(I) binding domain of ATP7B. Fig. 3(b) shows TM6, the sixth transmembrane domain of ATP7B modeled on M4, the fourth transmembrane domain of the Ca-ATPase. Val 304, Ile 307 and Glu 309 in M4, that contribute to site II high affinity Ca2+ ion binding site in SERCA1a [11], correspond to Cys 980, 983 and 985 in the unwound part of TM6, forming the CXXCPC motif. Site II is formed almost directly on M4 by the
Discussion
The difficulty in studying the entire ATP7B, due to its high molecular weight and poor solubility in aqueous media, necessitated the incorporation of CPC into a peptide model. The model study of CPC motif, part of Wilson TM6, by means of CD and 1H NMR spectroscopy, has revealed its significant interaction with Cu(I), implying a key role in copper transport.
The presence of CPC motif in both 2K8p and 1s results in significant conformation changes upon their interaction with Cu(I) as indicated by
Abbreviations
- BCA
bicinchoninic acid
- BSA
bovine serum albumin
- CT
charge transfer
- DMF
N,N-dimethyl-formamide
- DTT
dithiothreitol (2,3-dihydroxy-1,4-dithiobutane)
- ESI-MS
electrospray ionization mass spectroscopy
- Fmoc
9-fluorenyloxycarbonyl-
- GSH
glutathione
- HBTU
O-benzotriazolylo-tetramethylo-isourinate
- HPLC
high performance liquid chromatography
- LMCT
ligand to metal charge transfer
- NOESY
nuclear Overhauser effect spectroscopy
- PAL–PEG-PS
polysterene–polyethylene glycol polymer support
- ROESY
rotating Overhauser effect spectroscopy
- SDS
sodium
Acknowledgements
This work was supported by a grant (MOP-1800) from the Canadian Institute of Health Research.
References (49)
- et al.
Int. J. Biochem. Cell Biol.
(1998) J. Inorg. Biochem.
(2000)- et al.
J. Biol. Chem.
(2001) - et al.
Biophys. J.
(2001) - et al.
Am. J. Hum. Genet.
(1998) - et al.
J. Biol. Chem.
(1997) - et al.
J. Biol. Chem.
(2002) - et al.
Anal. Biochem.
(1985) - et al.
Anal. Biochem.
(1988) Arch. Biochem. Biophys.
(1959)
J. Biol. Chem.
J. Biol. Chem.
Nat. Genet.
Nat. Genet.
Nat. Genet.
Hum. Mol. Genet.
J. Biol. Chem.
Biochemistry
J. Am. Chem. Soc.
Nature
J. Exp. Biol.
Biochem. J.
Biosci. Biotechnol. Biochem.
Hum. Mol. Genet.
Cited by (15)
A review on adsorptive separation of toxic metals from aquatic system using biochar produced from agro-waste
2021, ChemosphereCitation Excerpt :The higher intake of copper in the food leads to endemic Tyrolean infantile cirrhosis and Indian childhood cirrhosis (Tanner et al., 1979; Muller et al., 1996). Hereditary metabolic issue provides Wilson's and Menkes' diseases (Walshe, 1995; Tumer et al., 1996; Myari et al., 2004). In addition, Cu has been related by implication with various neurological issue such as prion and Alzheimer's diseases along with bovine spongiform encephalopathy (Llanos and Mercer, 2002; Jomova and Valko, 2011).
Cu(II)-histones interaction related to toxicity-carcinogenesis
2014, Coordination Chemistry ReviewsCitation Excerpt :Indian childhood cirrhosis and endemic Tyrolean infantile, both result from high dietary levels of copper [8,9], whereas Menkes’ and Wilson's disease are due to genetic metabolic disorders [10,11]. Moreover, copper's status has also been associated indirectly with a number of neurological disorders, including Alzheimer's and prion diseases, including bovine spongiform encephalopathy (BSE) [12,13]. Cu(II) can bind to DNA bases efficiently [14,15] and cell exposure to high concentrations of Cu(II) lead to apoptosis [16,17] directly associated with oxidative stress [13,18–20].
The cadmium transport sites of CadA, the Cd<sup>2+</sup>-ATPase from Listeria monocytogenes
2006, Journal of Biological ChemistryUsing NMR spectroscopy to investigate the role played by copper in prion diseases
2020, Neurological SciencesWilson’s disease: A comprehensive review of the molecular mechanisms
2015, International Journal of Molecular SciencesUtilizing NMR and EPR spectroscopy to probe the role of copper in prion diseases
2013, Magnetic Resonance in Chemistry