Copper transfer to the N-terminal domain of the Wilson disease protein (ATP7B): X-ray absorption spectroscopy of reconstituted and chaperone-loaded metal binding domains and their interaction with exogenous ligands

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Abstract

The copper-transporting ATPases are 165–175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain. In ATP7B, the Wilson disease protein, the N-terminal domain is made up of six metal-binding sub-domains containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues. We have expressed the N-terminal domain of ATP7B as a soluble C-terminal fusion with the maltose binding protein. This expression system produces a protein which can be reconstituted with copper without recourse to the harsh denaturing conditions or low pH reported by other laboratories. Here we describe the reconstitution of the metal binding domains (MBD) with Cu(I) using a number of different protocols, including copper loading via the chaperone, Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. The results establish that the metal-binding domains bind Cu(I) predominantly in a bis cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. We have further observed that exogenous ligand binding induces the formation of a Cu–Cu interaction which may signal a conformational change of the N-terminal domain.

Introduction

Copper distribution in mammalian cells requires coordinated action of several proteins, including the oligomeric copper-uptake protein CTR1, the cytosolic metallochaperones CCS, Atox1, and Cox17, and the ATP-driven copper transporters ATP7A and ATP7B (also known as the Menkes protein (MNK) and the Wilson protein (WND), respectively) [1], [2]. The primary function of ATP7A and ATP7B is (i) to deliver copper into the secretory pathway, where copper can be incorporated into copper-dependent enzymes [3], [4], [5], and (ii) to export excess copper out of the cell [6], [7]. Mutations in the ATP7A and ATP7B proteins lead to disruption of normal copper distribution, leading to severe pathologies in human, known as Menkes disease and Wilson's disease, respectively [8].

The copper-transporting ATPases are 165–175 kDa membrane proteins, composed of 8 transmembrane segments and two large cytosolic domains, the N-terminal copper-binding domain and the catalytic ATP-hydrolyzing domain (Fig. 1). The N-terminal domain is made up of a number of metal-binding sub-domains (MBDs) containing the MXCXXC motif which is known to coordinate copper via the two cysteine residues [9], [10], [11], [12]. WND and MNK each contain six of these metal binding domains (MBD) while bacterial and yeast analogues generally contain only two. The catalytic ATP binding domain is located between trans-membrane helices six and seven. A putative inter-membrane metal coordination site corresponding to a CPC motif resides close to the ATP-binding domain in trans-membrane segment 6. Structure function relationships have been investigated by overexpression of MNK in CHO [13] and yeast cells [14], and WND in insect cells [15]. The structure–function relationships of the isolated N-terminal and ATP-binding domains have also been investigated via expression of each as soluble constructs [9], [10], [11], [16].

As part of our ongoing studies aimed at understanding the relationship between copper binding, catalytic activity, and trafficking, we are interested in understanding the coordination chemistry of the N-terminal domains in detail. It is likely that the function of the N-terminus depends at least in part on the structural consequences of copper binding to the subdomains, and on conformational changes which affect interdomain interactions [10], [17]. Solution structures for several single metal binding domains as well as the homologous copper chaperones have been published [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. Whereas the Hg(II) crystal structure of the Atx1 chaperone from S. cerevisiae contains a linear 2-coordinate Hg(II) center ligated to C15 and C18 [24], the S–Cu–S angles in the ATPases are estimated to be 115° and 132°, respectively, much less than the 180° value expected for linear 2-coordination. Three-coordination involving an exogenous thiol has been proposed as the origin of the third ligand since all potential protein-derived third ligands are at too long a distance to influence the Cu(I) coordination directly.

Since NMR does not detect copper coordination directly and is unable to address the origin of the putative third ligand, we have used X-ray absorption spectroscopy to probe the local coordination of the Cu(I) center. In earlier work we have shown that Cu(I) binds to the metallochaperone Atox1 in a linear bis-cysteinate coordination [29]. Our studies on a maltose-binding fusion of the N-terminal domain of Menkes protein also showed bis-cysteinate binding but with evidence of increased distortion from linearity [12]. Didonato and coworkers have published similar EXAFS results for WND [10], but Cobine et al. [30] report copper coordinations between 2 and 3-coordinate for MNK, with some evidence for a copper–copper interaction. It is unclear whether the observed differences originate from differences in fusion constructs used to isolate the soluble domains, methods of copper reconstitution, or differences in copper loading.

Two procedures for generation of the recombinant N-terminal domain of WND (N-WND) have been reported. DiDonato et al. [9], [10], [17] generated and expressed the N-terminal domain of the WND protein fused with the glutathione-s-transferase (GST). Although sufficient quantities could be generated using this approach, the protein was largely insoluble and had to be denatured and refolded before the analysis, complicating interpretation of the results. In our laboratory, we previously generated maltose-binding protein (MBP) fusions with N-WNDP or N-MNKP and co-expressed these proteins with thioredoxin encoded on a separate plasmid [11]. The significant advantage of the co-expression system is the ability to obtain N-WNDP and N-MNKP in a soluble form and to load these proteins with copper both in a cell and in vitro without recourse to harsh denaturation or low-pH.

Here we report the characterization of N-WND expressed as the soluble C-terminal maltose binding construct. We describe the reconstitution of the metal binding domains with Cu(I) using a number of different protocols, including copper loading via the chaperone Atox1. X-ray absorption spectra have been obtained on all these derivatives, and their ability to bind exogenous ligands has been assessed. This work establishes that the metal-binding domains of N-WND bind Cu(I) in a distorted bis-cysteinate environment, and are able to bind exogenous ligands such as DTT in a similar fashion to Atox1. An interesting new observation is that exogenous ligand binding induces the formation of a Cu–Cu interaction which may signal a conformational change of the N-terminal domain.

Section snippets

Overexpression and purification of N-WND – maltose binding protein fusions

The N-terminal domain of WND was overexpressed as a maltose binding protein fusion (pMAL-c2 vector, New England Biolabs) in Escherichia coli (BL 21) according to published procedures [11]. This recombinant domain termed N-WND-MBP corresponds to residues 1–612 fused to a C-terminal maltose binding protein. Co-expression of N-WND with thioredoxin prevented the cysteine residues from forming disulfide bridges with subsequent misfolding and formation of inclusion bodies. In a typical experiment,

Copper reconstitution

Expression of N-WND as a C-terminal maltose binding fusion protein produced a soluble protein with a yield of about 5 mg per liter of culture. As isolated, the protein contained variable amounts of copper. It could be reconstituted by a variety of protocols. If copper (as cupric ion) was added to the cell culture prior to induction, the metal was incorporated into N-WND-MBP as Cu(I) to generate Cu/P ratios which varied depending on the concentration of Cu(II) added to the medium. When no copper

Acknowledgements

The work was supported by a Program Project Grant P01-GM067166 to N.J.B. and S.L. We thank Deepali Datta for assistance in preparation of HAH1. We thank Dr James E. Penner-Hahn for making available the XAS data on the bis-tetramethylbenzenethiolate–Cu(I) model complex, and Dr Graham George for making available the XAS data on the tris-tetramethylthiourea–Cu(I) complex. We gratefully acknowledge the use of facilities at the Stanford Synchrotron Radiation Laboratory, which is supported by the

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