A comparative structural genomic analysis of a new class of metal-trafficking proteins can provide insights into the intracellular chemistry of reactive cofactors such as copper and zinc. Starting from the sequences of the metallochaperone Atx1 and from the first soluble domain of the copper-transporting ATPase Ccc2, both from yeast, a search on the available genomes was performed using a homology criterion and a metal-binding motif x‘-x"-C-x‴-x⁗-C. By limiting ourselves to 20% identity with any of the proteins found, several soluble copper-transport proteins were identified, as well as soluble domains of membrane-bound ATPases. Structural models were calculated using high-resolution solution structures as templates, and the models were validated using statistical and energy criteria. Residue conservation and substitution have been interpreted and discussed in terms of structure–function relationship. The potential energy surfaces have been analyzed in terms of protein–protein interactions. We find that metallochaperones and their physiological partner ATPases from several phylogenetic kingdoms recognize one another, via an interplay of electrostatics, hydrogen bonding, and hydrophobic interactions, in a manner that precisely orients the metal-binding side chains for rapid metal transfer between otherwise tight binding sites. Finally, other putative metal-transport proteins are mentioned that have low homology and/or a different metal-binding consensus motif and that appear to use similar structures for recognition and transfer. This analysis highlights the wealth and the complexity of the field.

The eukaryotic copper,zinc superoxide dismutases are remarkably stable dimeric proteins that maintain an intrasubunit disulfide bond in the reducing environment of the cytosol and are active under a variety of stringent denaturing conditions. The structural interplay of conserved disulfide bond and metal-site occupancy in human copper,zinc superoxide dismutase (hSOD1) is of increasing interest as these post-translational modifications are known to dramatically alter the catalytic chemistry, the subcellular localization, and the susceptibility of the protein to aggregation. Using biophysical methods, we find no significant change in the gross secondary or tertiary structure of the demetallated form upon reduction of the disulfide. Interestingly, reduction does lead to a dramatic change in the quaternary structure, decreasing the monomer-to-dimer equilibrium constant by at least four orders of magnitude. This reduced form of hSOD1 is monomeric, even at concentrations well above the physiological range. Either the addition of Zn(II) or the formation of the disulfide leads to a shift in equilibrium that favors the dimeric species, even at low protein concentrations (i.e. micromolar range). We conclude that only the most immature form of hSOD1, i.e. one without any post-translational modifications, favors the monomeric state under physiological conditions. This finding provides a basis for understanding the selectivity of mitochondrial SOD1 import and may be relevant to the toxic properties of mutant forms of hSOD1 that can cause the familial form of amyotrophic lateral sclerosis.

The protein CopC from Pseudomonas syringae has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously. The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91. A full solution structure analysis is reported here for Cu(I)-CopC. The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region. Both copper binding sites have been characterized through extended x-ray absorption fine structure studies. They represent novel coordination environments for copper in proteins. The two sites are ≈30 Å far apart and have little affinity for the ion in the other oxidation state. Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other. This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch. CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper. CopC and CopA are coded in the same operon, responsible for copper resistance. These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis.

The interaction of the copper chaperone Atx1 and the first cytosolic domain of Ccc2 ATPase, Ccc2a, was investigated by NMR in solution. In particular, a solution of Cu(I)-¹⁵NAtx1 was titrated with apo-Ccc2a, and, vice versa, a solution of Cu(I)-¹⁵NCcc2a was titrated with apo-Atx1. By following the ¹⁵N and ¹H chemical shifts, a new species is detected in both experiments. This species is the same in both titrations and is in fast exchange with the parent species on the NMR time scale. Nuclear relaxation data are consistent with the formation of an adduct. Judging from the nuclear Overhauser effect spectroscopy patterns, the structure of Cu(I)-¹⁵NCcc2a in the presence of apo-Atx1 is not significantly altered, whereas Cu(I)-¹⁵NAtx1 in the presence of apo-Ccc2a experiences some changes with respect to both the apoproteins and the Cu(I)-loaded proteins. The structure of the Cu(I)-¹⁵NAtx1 moiety in the adduct was obtained from 1137 nuclear Overhauser effects to a final root mean square deviation to the mean structure of 0.76 ± 0.13 Å for the backbone and 1.11 ± 0.11 Å for the heavy atoms. ¹⁵N and ¹H chemical shifts suggest the regions of interaction that, together with independent information, allow a structural model of the adduct to be proposed. The apo form of Atx1 displays significant mobility in loops 1 and 5, the N-terminal part of helix α₁, and the C-terminal part of helix α₂ on the ms–μs time scale. These regions correspond to the metal binding site. Such mobility is largely reduced in the free Cu(I)-Atx1 and in the adduct with apo-Ccc2a. The analogous mobility of Ccc2a in both Cu(I) and apo forms is reduced with respect to Atx1. Such an adduct is relevant as a structural and kinetic model for copper transfer from Atx1 to Ccc2a in physiological conditions.