Copper-related dysfunction can arise from either copper deficiency or copper overload. The essential, yet toxic nature of copper is highlighted in the genetic disorders Menkes and Wilson disease. Symptoms of copper overload include liver failure and neurodegeneration, while copper deficiency results in a loss of activity of cuproenzymes important in respiratory pathway, connective tissue formation and stress responses. Regulation of intracellular copper requires that the proteins involved respond rapidly to changes in copper concentrations. Metal-binding is an important trigger for activity, the metal-binding properties of the individual proteins are critical to understanding the balance required in copper metabolism. The utility and extension of these studies is evident by the conservation of copper homeostasis proteins from prokaryotes to eukaryotes.
The Menkes ATPase (MNK) is critical to copper homeostasis in humans. Mutations in the gene encoding the ATPase result in a fatal disorder related to copper deficiency. Copper has been shown previously to regulate the activity and location of MNK. The response to extracellular copper is rapid and has been proposed to proceed as a result of metal binding to the amino-terminal domain. Each of the six amino-terminal domains (MNKr) has a CxxC motif proposed to bind copper. Spectroscopic evidence indicates that MNKr binds copper(I) and silver(I) but not cadmium(II) and zinc(II). UV-visible spectroscopy of MNKr titrated with copper yielded an increasing Cu(I)-S metal-ligand charge transfer band at approximately 250 nm. Additionally the UV-visible spectrum contained no evidence of Cu(II) transitions. X-ray absorption studies confirmed the cuprous reduction state and suggested the probable ligands as sulfurs. Cu(I)-MNKr has luminescence properties consistent with a Cu(I)-S coordination in an environment that is shielded from solvent interactions. The combination of UV-visible and luminescence spectroscopy has suggested a stoichiometry of four Cu(I) ions per protein. X-ray absorption data supports the formation of a Cu(I)-S cluster with sharing of sulfurs between the six proposed metal-binding motifs with bond lengths indicative of two- and three-coordinate copper (2.20Å) with a proximal metal-metal interaction at 2.69Å. The hypothesis is that the folding of the domains into a conformation that allows for the luminescence and metal-metal interaction plays an important role in the regulation of the ATPase activity and location.
The copper chaperones have been identified as the proteins that deliver copper through the cellular milieu to specific targets. These proteins allow the cell to protect itself against the deleterious effects of free copper but still allow activation of copperdependent enzymes. The Gram-positive bacteria Enterococcus hirae has one identified copper chaperone, CopZ, which is part of the βαββαβ folded family of proteins that bind Cu(I) with exposed cysteinyl sulfurs. CopZ acts as a copper chaperone delivering copper via a specific interaction with cop operon repressor, CopY. UV-visible spectroscopy of CopZ demonstrates a stoichiometry of one Cu(I) per CopZ and XAS studies suggest that the copper is in a two-coordinate sulfur site with bond lengths of 2.24Å. The metal-coordinating sulfurs are exposed on a loop between the first β-strand and first a-helix. The site is exposed and flexible allowing for copper transfer. Copper transfer from CopZ to the repressor CopY via an electrostatic mediated interaction results in a luminescent Cu(I)-S core being formed in CopY. The stoichiometry of transfer indicates that multiple CopZ molecules interact to displace zinc from CopY and disrupt DNA-binding. The zinc coordinated in CopY plays a role in the DNA-binding and in the protein-protein interaction required to regulate the activity of CopY. Zn(II) is coordinated in tetrahedral coordination with four sulfur ligands at 2.35Å. The zinc is displaced when CopY forms a binuclear copper-thiolate cluster with two three coordinate Cu(I) ions with average Cu(I)-S bonds of 2.26Å and a proximal Cu(I) at 2.69Å.
The CopZ-CopY interaction is specific. An individual Menkes sub-domain MNKr2, has an identical global fold and metal-binding properties yet is unable to transfer copper to CopY. Although the βαββαβ fold provides a similar exposed CxxC metal-binding loop with a two-coordinate Cu(I) site, the electrostatic surface potentials are quite different. The interaction of CopY and CopZ can be mimicked by the inclusion of a charge surface on MNKr2 that more closely resembles the surface of CopZ. The addition of a pair of lysine residues positioned proximal to the metal-binding site confers the ability to transfer copper. These gain-of-function interactions have been used to investigate stoichiometry of the interaction and potential conformational changes involved in copper transfer.
The role of zinc in the CopY dimer has been investigated using a truncation of CopY, Ymbs. The metal-binding characteristics of Ymbs are equivalent to CopY in terms of stoichiometry and metal-induced dimerization. X-ray absorption studies demonstrate two three-coordinate Cu(I) displaces a tetrahedral Zn(II) (Zn(II) -S bond lengths of2.35Å). The average Cu(I)-S bond lengths of 2.24Å and Cu-Cu scatter at 2.66Å correlate with the data collected for CopY. The truncate is unable to bind DNA but suggests that zinc mediates a structure that enhances the protein-protein interactions and helps mediate the DNA-binding activity. Nuclear magnetic resonance data suggest binding of zinc orders a structure that is changed by the displacement of zinc. The conformational change in this domain affects the activity of the repressor.
Neurospora crassa metallothionein, NcMT, is a copper-specific metallothionein. However, the in vitro copper-binding stoichiometry is mediated by zinc binding to apoNcMT. The copper-binding stoichiometry of apoNcMT appears to be 4 Cu(I) ions per NcMT while titration with zinc results in stoichiometry of 6 Cu(I) ions per NcMT. The 6 Cu(I)NcMT has a homogenous conformation while the 4 Cu(I) species appears to be mixture of conformations. Zinc-binding prior to the Cu(I) titration presumably limits the conformations adopted during the titration. This flexibility has been a significant factor that has prevented the solution of other in vitro prepared Cu(I)-MT structures. The NcMT and CopY data suggest an important role for zinc in stabilizing a fold that affects copper-binding characteristics.