Crystal structure of human copper homeostasis protein CutC reveals a potential copper-binding site
Introduction
Copper is a trace metal element essential to all organisms which can act as a cofactor for many enzymes involved in redox reactions and various biological processes, including electron transfer in cytochrome, neuropeptides modification, and detoxification by Cu–Zn superoxide dismutase, etc. (Lee et al., 2001, Rensing and Grass, 2003). Copper homeostasis is strictly and precisely controlled to prevent copper accumulation beyond cellular needs because excessive copper is highly toxic to cells and can lead to cell death through binding to some essential cellular components (Pena et al., 1999). Disturbed copper homeostasis has been implicated in several genetic diseases in both human beings and animals including Menkes disease and Wilson disease (DiDonato and Sarkar, 1997, Schaefer and Gitlin, 1999).
So far, several gene families have been identified to be responsible for maintaining the balance of copper in cells. In bacteria, two gene families are involved in copper homeostasis from uptake to storage, delivery, and efflux (reviewed in Silver and Phung, 1996). One of them, the cop gene family, is well characterized and associated with uptake and export of copper. The other one, the cut gene family, is defined based on a preliminary characterization of copper-sensitive mutants. The cut gene family contains six members (cutA, cutB, cutC, cutD, cutE, and cutF) and has also been suggested to be involved in uptake, intracellular storage and transport, and efflux of copper. Among the cut members, cutC is conserved from bacteria to mammals. Mutation (Gupta et al., 1995) or deletion (Rensing and Grass, 2003) of cutC in Escherichia coli leads to increased copper sensitivity at high concentration of copper, probably due to a decreased efflux of copper (Gupta et al., 1995). Those results suggest that CutC may play an important role in intracellular trafficking of Cu(I) to alleviate the stress (Kimura and Nishioka, 1997, Rensing and Grass, 2003). In Caenorhabditis elegans, knockdown of cutC also increases the copper sensitivity and affects the worm phenotype at high level of copper (Calafato et al., 2008). To date, only the structure of CutC from Shigella flexneri (sfCutC) is described, which adopts a classical TIM-barrel fold and represents a new structural family of TIM-barrel (Zhu et al., 2005). However, the structure of sfCutC reveals very limited information about the biological function of CutC.
Human CutC (hCutC) is a cytoplasmic protein of 273 amino acid residues with a molecular mass of 30 kDa and is expressed ubiquitously in nearly all human tissues (Li et al., 2005, Ota et al., 2004). To gain insights into the potential function of hCutC, we carried out biochemical studies of hCutC and determined the crystal structure of hCutC at 2.5 Å resolution. The biochemical and structural data together suggest that hCutC is a Cu(I)-binding protein with a stoichiometry of 1:1 and an apparent dissociation constant of 15.5 ± 2.8 μM, and the potential Cu(I)-binding site consists of two strictly conserved Cys residues and other conserved residues nearby on the inner surface of the C-terminal end of the TIM-barrel. These results provide useful information for further functional analysis of hCutC.
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Cloning, expression, and purification
The hCutC gene (GenBank Accession No. NP_057044) was amplified by PCR from the cDNA library of human CD34 + haematopoietic stem/progenitor cells (Mao et al., 1998) and inserted into the SphI and PstI restriction sites of the expression vector pQE30 (Qiagen) which attaches a hexa-His tag at the N-terminus of the target protein. Plasmids encoding all mutant versions of hCutC were generated by site-directed mutagenesis using QuikChange kit (Stratagene). The wild-type hCutC in pQE30 vector was used
Copper-binding analysis of hCutC
The previous biological data have suggested that CutC may play an important role in Cu(I) trafficking (Kimura and Nishioka, 1997, Rensing and Grass, 2003). To elucidate the biological function of hCutC, we carried out a biochemical assay on the binding ability of hCutC with Cu(I). The biochemical results show that hCutC is able to bind Cu(I) in vitro with a stoichiometry of 1:1 (Fig. 1C), suggesting that hCutC has one Cu-binding site per molecule. The apparent dissociation constant (Kd) was
Conclusion
CutC is a family of proteins conserved from bacteria to mammals that have been implicated to play an important role in the intracellular trafficking of Cu(I). However, the exact biological function of CutC is unclear. We have analyzed the copper-binding ability and determined the crystal structure of hCutC. The biochemical data show that hCutC can bind Cu(I) with a stoichiometry of 1:1 and a relatively low binding affinity (Kd = 15.5 ± 2.8 μM). hCutC adopts a typical TIM-barrel fold and exists as a
Acknowledgments
We thank the staff members at Photon Factory, Japan for support in diffraction data collection and other members of our group for helpful discussion. This work was supported by grants from the Ministry of Science and Technology of China (2006AA02A313 and 2007CB914302), the National Natural Science Foundation of China (30730028), the Chinese Academy of Sciences (KSCX2-YW-R-107 and SIBS2008002), and the Science and Technology Commission of Shanghai Municipality (07XD14032).
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