Structure and metal binding studies of the second copper binding domain of the Menkes ATPase

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Abstract

Biological utilisation of copper requires that the metal, in its ionic forms, be meticulously transported, inserted into enzymes and regulatory proteins, and excess be excreted. To understand the trafficking process, it is crucial that the structures of the proteins involved in the varied processes be resolved. To investigate copper binding to a family of structurally related copper-binding proteins, we have characterised the second Menkes N-terminal domain (MNKr2). The structure, determined using 1H and 15N heteronuclear NMR, of the reduced form of MNKr2 has revealed two α-helices lying over a single β-sheet and shows that the binding site, a Cys(X)2Cys pair, is located on an exposed loop. 1H–15N HSQC experiments demonstrate that binding of Cu(I) causes changes that are localised to conserved residues adjacent to the metal binding site. Residues in this area are important to the delivery of copper by the structurally related Cu(I) chaperones. Complementary site-directed mutagenesis of the adjacent residues has been used to probe the structural roles of conserved residues.

Introduction

The essential nature of copper is illustrated by the human X-linked genetic disorder known as Menkes disease (Bull and Cox, 1994). Copper is used as a catalytic or structural cofactor in a variety of critical enzymes and proteins. Menkes disease is an early childhood disease characterised by neurological problems and connective tissue abnormalities resulting from copper deficiency. The gene defective in Menkes disease, ATP7A, encodes a copper transporting CPx-type ATPase (Vulpe et al., 1993). The Menkes ATPase transports copper into the trans-Golgi for eventual uptake into nascent cuproenzymes and is also involved in detoxification of copper. The ATPase equilibrates between the trans-Golgi network and the plasma membrane and in conditions of excess copper the equilibrium favours the plasma membrane (Petris et al., 1996).

The CPx-type ATPases have a conserved cysteine and proline (CPx—x is either cysteine or histidine) motif, and between one and six N-terminal metal binding domains (Solioz and Vulpe, 1996). The Menkes ATPase has six such domains, each approximately 70 amino acids in length and containing a single –Cys(X)2Cys– (X is any amino acid) metal binding motif. Proteins with this motif, termed copper chaperones, are also found transporting copper(I) intracellularly. The Menkes domains are tethered together by polypeptide sequences that have no sequence or length similarities but may provide structural elements or modulate motions of discrete modules (Arnesano et al., 2002). Mutation of the metal binding cysteine residues has shown that copper binding to the N-terminal domains is necessary for ATPase redistribution to the plasma membrane, but also indicated that not all six modules are necessary for correct copper trafficking (Voskoboinik et al., 1999). The N-terminal domains of the Wilson ATPase, a Menkes homologue, have been shown to interact with the ATP binding domain in a copper dependent manner (Tsivkovskii et al., 2001). Further studies with the Wilson domains show that the matallochaperone Hah1 interacts with some of the domains and that the six domains do not have equivalent functions (Hamza et al., 1999; Larin et al., 1999; Walker et al., 2002). These results suggest that the N-terminal domains play an important role in regulating the activity of the ATPase. Further, the six Menkes N-terminal domains have been proposed to bind four Cu(I) ions in a solvent shielded cluster (Cobine et al., 2000). To achieve such a conformation, the six domains must have some interaction with each other. Clearly, determining the structure of multiple copper binding domains provides an important first step in attempting to understand copper binding and protein–protein interactions necessary for correct biological function.

The NMR structure of the fourth domain in both the apo- and Ag(I) loaded states (Gitschier et al., 1998) shows that the domain adopts a fold known as an “open-faced β-sandwich” (Richardson, 1981). The first domain of a yeast copper trafficking ATPase also displays this fold (Banci et al., 2001), which indicates that the N-terminal domains of copper trafficking ATPases from different organisms have evolved with similar structures. An ATPase from Bacillus subtilis, which has two –Cys(X)2Cys– motif containing N-terminal domains, has recently been characterised. Interestingly, of the two domains only one is structured in the apo- and Cu(I)-loaded states (Banci et al., 2002). The unstructured portion of BsCopA may suggest that although the Menkes ATPase has six N-terminal domains identified in the primary sequence not all domains may be structured.

The N-terminal domains of the CPx-type ATPases and copper chaperones share homology and conserved structural features (Arnesano et al., 2002; Huffman and O’Halloran, 2001). In the yeast copper chaperone, Atx1, a lysine, important for correct function, is conserved on a loop adjacent to the copper-binding site (Portnoy et al., 1999). The positive side-chain of the lysine residue is thought to be involved in stabilising the copper thiolate centre (Arnesano et al., 2001b). Rather than a lysine residue many eukaryotic ATPase domains have a conserved phenylalanine. Bulky, aromatic residues in and around metal binding sites have been proposed to influence metal binding affinity by reducing the conformational flexibility of the metal ligands (Hunt et al., 1999). Additionally, the hydrophobic nature of aromatic residues may influence metal binding affinity by altering the electrostatic interactions within the binding site (Yamashita et al., 1990). Hence, the conserved phenylalanine residue may help stabilise the metal binding site in an electrostatic fashion. Indeed, the mobility of the yeast Menkes homologue (Ccc2a), which has a phenylalanine, is less than its chaperone (Atx1), which utilises a lysine (Arnesano et al., 2001a). Possibly, this difference is due to the conserved phenylalanine stabilising the metal binding site.

To further our investigations into copper binding by the Menkes domains and mechanisms of copper transport and delivery we have determined the structure of apo-MNKr2 and have analysed changes that occur upon copper binding. We show that the module forms a global fold with a β1α1β2β3α2β4 arrangement of secondary structural elements. Copper induced structural changes identified from chemical shift perturbation of MNKr2 backbone 1HN and 15N resonances are localised to residues in the vicinity of the metal binding site, similar to that found when the fourth domain bound silver. Furthermore, a mutant was prepared to investigate the role of the conserved phenylalanine residue (Phe72) in influencing the copper binding ability of MNKr2.

Section snippets

Protein expression and purification

The sequence encoding MNKr2 was cloned into pET29-a (Novagen) and transformed into Escherichia coli BL21(DE3) cells. The MNKr2 sequence was confirmed by automated dideoxynucleotide sequencing. To prepare unlabelled MNKr2, transformed cells were grown in LB media containing 35 μg/L kanamycin sulfate at 37 °C and induced at A595=0.8 with isopropyl-β-d-thiogalactopyranoside for 3 h. The cells were harvested by centrifugation at 4000g for 20 min at 4 °C, resuspended in buffer A (100 mM potassium

Spin system assignment and description of secondary structure of apo-MNKr2

The solution structure of apo-MNKr2 was determined using standard homonuclear and heteronuclear NMR methods (Wuthrich, 1986). The spin systems were determined from 298 K TOCSY and DQF-COSY spectra with reference to a 3D 15N TOCSY-HSQC spectrum where overlap occurred in the homonuclear spectra. A 298 K NOESY (τm=150 ms) spectrum was used to determine sequential connectivities and identify long-range contacts. Where ambiguity of a particular NOE was encountered, reference was made to either a 3D 15N

Structure of MNKr2

The structure of MNKr2 indicates that the protein is a member of a family of Cu(I) binding proteins that includes the fourth domain of the Menkes ATPase (Gitschier et al., 1998), the first domain of a yeast ATPase, Ccc2p (Banci et al., 2001), a domain of the B. subtilis ATPase, BsCopA(73–147) (Banci et al., 2002), and some copper metallochaperones (Huffman and O’Halloran, 2001). The Cys(X)2Cys metal binding motif is located on the loop connecting the first β-strand with the first helix. This

Acknowledgements

We thank Dr. Martin Scanlon for assistance with the recording of preliminary NMR spectra. CEJ is the recipient of an Australian National Health and Medical Research Council ‘Dora Lush’ postgraduate scholarship and wishes to acknowledge the support of the Alfred and Olivea Wynne Trust. This work was supported by a grant to CTD from the Australian Research Council. DJC acknowledges the support of an Australian Research Council Professorial Fellowship. The Institute for Molecular Biosciences is a

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