Copper(I) interaction with model peptides of WD6 and TM6 domains of Wilson ATPase: regulatory and mechanistic implications

https://doi.org/10.1016/j.jinorgbio.2004.05.013Get rights and content

Abstract

With the aim to investigate the mechanism of Cu(I) transport by Wilson ATPase (ATP7B), we have studied the interaction of the peptides 2K10p (CH3CO–Lys–Gly–Met–Thr–Cys–Ala–Ser–Cys–Val–His–Asn–Lys–CONH2), and 2K8p (CH3CO–Lys–Leu–Cys–Ile–Ala–Cys–Pro–Cys–Ser–Lys–CONH2), part of the sixth metal binding domain (WD6) and the sixth transmembrane segment (TM6) of Wilson ATPase, respectively, by means of CD, NMR spectroscopy and homology modeling. In addition, the interaction of Cu(I) with the 2K8p mutants 1s (CH3CO–Lys–Leu–Ser–Ile–Ala–Cys–Pro–Cys–Ser–Lys–CONH2), 2s (CH3CO–Lys–Leu–Cys–Ile–Ala–Ser–Pro–Cys–Ser–Lys–CONH2) and 3s (CH3CO–Lys–Leu–Cys–Ile–Ala–Cys–Pro–Ser–Ser–Lys–CONH2), containing two cysteines in various positions, have been studied with the same methods, in order to understand the role of each cysteine in copper binding. Our studies show that the three cysteine thiolates present in the 2K8p peptide sequence act mainly as bridging ligands for Cu(I) binding, and dithiothreitol acts as an important ligand in Cu(I) ligation by 2K10p and the 2K8p mutants. Formation of oligomeric species has been evidenced for all peptides except 2s. Shift of the equilibrium between the various oligomeric species has been accomplished by reducing the Cu(I):peptide ratio. Significant shifts of proline protons upon interaction with Cu(I) have been observed for all proline containing peptides implying a possible role of proline in facilitating Cu(I) binding. These findings have been further discussed with respect to the molecular basis of copper trafficking and intermolecular interactions.

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)

  • K. Terada et al.

    Int. J. Biochem. Cell Biol.

    (1998)
  • B. Sarkar

    J. Inorg. Biochem.

    (2000)
  • Z. Hou et al.

    J. Biol. Chem.

    (2001)
  • W.J. Rice et al.

    Biophys. J.

    (2001)
  • J.R. Forbes et al.

    Am. J. Hum. Genet.

    (1998)
  • I.H. Hung et al.

    J. Biol. Chem.

    (1997)
  • M.J. Petris et al.

    J. Biol. Chem.

    (2002)
  • P.K. Smith et al.

    Anal. Biochem.

    (1985)
  • K. Wiechelman et al.

    Anal. Biochem.

    (1988)
  • G.L. Ellman

    Arch. Biochem. Biophys.

    (1959)
  • S.C. Li et al.

    J. Biol. Chem.

    (1993)
  • R. Wimmer et al.

    J. Biol. Chem.

    (1999)
  • K. Petrukhin et al.

    Nat. Genet.

    (1993)
  • R.E. Tanzi et al.

    Nat. Genet.

    (1993)
  • P.C. Bull et al.

    Nat. Genet.

    (1993)
  • J.R. Forbes et al.

    Hum. Mol. Genet.

    (2000)
  • J.R. Forbes et al.

    J. Biol. Chem.

    (1989)
  • R.A. Steele et al.

    Biochemistry

    (1997)
  • G. Veglia et al.

    J. Am. Chem. Soc.

    (2000)
  • C. Toyoshima et al.

    Nature

    (2000)
  • G.A. Scarborough

    J. Exp. Biol.

    (2000)
  • K.J. Sweadner et al.

    Biochem. J.

    (2001)
  • T. Yoshimizu et al.

    Biosci. Biotechnol. Biochem.

    (1998)
  • J.R. Forbes et al.

    Hum. Mol. Genet.

    (2000)
  • Cited by (15)

    • A review on adsorptive separation of toxic metals from aquatic system using biochar produced from agro-waste

      2021, Chemosphere
      Citation 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 Reviews
      Citation 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].

    • Wilson’s disease: A comprehensive review of the molecular mechanisms

      2015, International Journal of Molecular Sciences
    View all citing articles on Scopus
    View full text