Characterization and Structure of a Zn2+ and [2Fe-2S]-containing Copper Chaperone from Archaeoglobus fulgidus

Matthew H Sazinsky,Benjamin Lemoine,Maria Orofino,Roman Davydov,Krisztina Z Bencze,Timothy L Stemmler,Brian M Hoffman,José M Argüello,Amy C Rosenzweig

doi:10.1074/jbc.m703311200

Abstract

Bacterial CopZ proteins deliver copper to P1B-type Cu+-ATPases that are homologous to the human Wilson and Menkes disease proteins. The genome of the hyperthermophile Archaeoglobus fulgidus encodes a putative CopZ copper chaperone that contains an unusual cysteine-rich N-terminal domain of 130 amino acids in addition to a C-terminal copper binding domain with a conserved CXXC motif. The N-terminal domain (CopZ-NT) is homologous to proteins found only in extremophiles and is the only such protein that is fused to a copper chaperone. Surprisingly, optical, electron paramagnetic resonance, and x-ray absorption spectroscopic data indicate the presence of a [2Fe-2S] cluster in CopZ-NT. The intact CopZ protein binds two copper ions, one in each domain. The 1.8 A resolution crystal structure of CopZ-NT reveals that the [2Fe-2S] cluster is housed within a novel fold and that the protein also binds a zinc ion at a four-cysteine site. CopZ can deliver Cu+ to the A. fulgidus CopA N-terminal metal binding domain and is capable of reducing Cu2+ to Cu+. This unique fusion of a redox-active domain with a CXXC-containing copper chaperone domain is relevant to the evolution of copper homeostatic mechanisms and suggests new models for copper trafficking.

Highlights

Copper is a meticulously regulated redox-active micronutrient found in a number of important enzymes, including cytochrome c oxidase and superoxide dismutase
Both disorders are caused by mutations in Cuϩtransporting P1B-type ATPases [5,6,7], enzymes that are found in most organisms and function in the cellular localization and/or export of cytosolic copper [8, 9]
Each metal binding domains (MBDs) contains a highly conserved CXXC consensus sequence for binding Cuϩ and adopts a ␤␣␤␤␣␤ fold [11,12,13,14] nearly identical to that of the Atx1-like cytosolic copper chaperones, including yeast Atx1, human Atox1, and bacterial CopZ [15,16,17,18,19]. These chaperones contain a CXXC motif and deliver Cuϩ to one or all of the MBDs (20 –26). It is not clear how Cuϩ reaches the transmembrane metal-binding site and how the cytosolic chaperones participate in this process

Summary

MATERIALS AND METHODS

Cloning and Purification of CopZ and the CopA N-terminal MBD—The gene encoding CopZ (AF03456, GenBankTM accession number NP_069182) was cloned from A. fulgidus genomic DNA by PCR using the primers 5Ј-ATGATGCGATGCCCAGAATG-3Ј and 5Ј-TCTCTTTCAAGCCGTGCAGA3Ј. Full-length CopZ was expressed as described above in cells grown in minimal media supplemented with 100 ␮M iron ammonium sulfate that contained less than 10 ␮M zinc. A small sample of 2 mM CopZ in 25 mM Tris, pH 7.5, 100 mM NaCl, 5% glycerol was frozen at 100 K on a standard protein crystal mounting loop and exposed to x-rays tuned to the absorption edges of iron, cobalt, nickel, copper, zinc, and tungsten. Cuϩ was added in 6.6-fold excess to the column containing bound CopA N-MBD and incubated for 10 min at room temperature to initiate copper exchange. The root mean square difference (r.m.s.d.) for backbone atoms between the two molecules in the asymmetric unit is 0.3 Å, and no significant structural differences were observed

RESULTS AND DISCUSSION

APS beamline

Ligand environmenta

Anomalous peak heights at the iron and selenium absorption edges