Copper uptake proteins (CTRs), mediate cellular acquisition of the essential metal copper in all eukaryotes. Here, we report the structure of the human CTR1 protein solved by electron crystallography to an in plane resolution of 7 Å. Reminiscent of the design of traditional ion channels, trimeric hCTR1 creates a pore that stretches across the membrane bilayer at the interface between the subunits. Assignment of the helices identifies the second transmembrane helix as the key element lining the pore, and reveals how functionally important residues on this helix could participate in Cu(I)-coordination during transport. Aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCTR1 appear to provide additional metal binding sites. Consistent with the existence of distinct metal binding sites, we demonstrate that hCTR1 stably binds 2 Cu(I)-ions through 3-coordinate Cu-S bonds, and that mutations in one of these putative binding sites results in a change of coordination chemistry.copper homeostasis ͉ electron crystallography ͉ EXAFS ͉ membrane protein

Extensive evidence points to oxidative stress as a key event in the pathogenesis and exacerbation of Alzheimer's Disease (AD).[1] Transition metals, such as Zn, Fe, and Cu, are present in elevated concentrations in AD brain deposits, composed primarily of 40-or 42-mer amyloid beta (Aβ) peptides. The redox-active copper(II) ion binds to the unstructured, hydrophilic N terminus of Aβ; [1g,2] and the ability of copper to promote the formation of reactive oxygen species (ROS) and cause neuronal death by interaction with Aβ has been demonstrated in vitro. [1a,c,3,4] ROS formation is proposed to occur by interaction of reduced Cu I -Aβ with O 2 or H 2 O 2 . However, few direct studies of Cu I binding or reactivity with Aβ peptides or fragments have been reported. [5,6] We have studied the interactions of the hydrophilic N-terminal region of the Aβ peptide with Cu I . An understanding of the full redox competency of Cu-Aβ, leading to ROS formation and oxidative stress (that is, to cause events associated with the onset of AD), is incomplete without elucidation of the structure/function relationships of the reduced (active) copper(I)-peptide complexes. We report herein studies on the interaction of Cu I ions with small portions of the Aβ peptide incorporating specific metal-binding (His6, His13, His14) or potentially redoxactive (Tyr10) residues (Figure 1). Of considerable interest are the contiguous His13 and His14 residues. We have previously reported studies on Cu I complexes of modified (by end-capping and/or regiospecific N ε -or N δ -alkylation) His-His dipeptides which, significantly, adopt a two-coordinate, near-linear N His -Cu I -N His environment. [6] In this report, we demonstrate that Cu I complexes of longer Aβ peptide fragments adopt the same apparent two-coordinate structure in the solid state and aqueous solution. Preliminary reactivity investigations, described here, indicate that the His13-Cu I -His14 moiety is the active part of the structure, responsible for copper-Aβ reactivity.A range of peptides ( Figure 1) were synthesized and purified by reverse-phase (RP) HPLC to a single peak. Their identity and purity were confirmed by ESI mass spectrometry. The peptides

Sco is a mononuclear red copper protein involved in the assembly of cytochrome c oxidase. It is spectroscopically similar to red copper nitrosocyanin, but unlike the latter, which has one copper cysteine thiolate, the former has two. In addition to the two cysteine ligands (C45 and C49), the WT protein from Bacillus subtilis (hereafter named BSco) has a histidine (H135) and an unknown endogenous protein oxygen ligand in a distorted tetragonal array. We have compared the properties of the WT protein to variants in which each of the two coordinating Cys residues has been individually mutated to Ala, using UV/vis, Cu and S K edge XAS, EPR, and resonance Raman spectroscopy. Unlike the Cu(II) form of native Sco, the Cu(II) complexes of the Cys variants are unstable. The copper center of C49A undergoes autoreduction to the Cu(I) form which is shown by EXAFS to be composed of a novel 2-coordinate center with one Cys and one His ligand. C45A rearranges to a new stable Cu(II) species coordinated by C49 H135 and a second His ligand recruited from a previously uncoordinated protein side chain. The different chemistry exhibited by the Cys variants can be rationalized by whether a stable Cu(I) species can be formed by autoredox chemistry. For C49A, the remaining Cys and His residues are trans which facilitate the formation of the highly stable 2-coordinate Cu(I) species, while for C45A such a configuration cannot be attained. Resonance Raman spectroscopy of the WT protein indicates a net weak Cu-S bond strength at ~ 2.24 Å corresponding to the two thiolate copper bonds, whereas the single variant C45A shows a moderately strong Cu-S bond at ~ 2.16 Å. S K-edge data gives a total covalency of 28% for both Cu-S bonds in the WT protein. These data suggest an average covalency per Cu-S bond lower than nitrosocyanin and close to that expected for type-2 Cu(II)-thiolate systems. The data are discussed relative to the unique Cu-S characteristics of cupredoxins, whence it is concluded that Sco does not contain highly covalent Cu-S bonds of the type expected for long-range electron transfer reactivity.

Copper binding and X-ray aborption spectroscopy studies are reported on untagged human CCS (hCCS; CCS = copper chaperone for superoxide dismutase) isolated using an intein self-cleaving vector and on single and double Cys to Ala mutants of the hCCS MTCQSC and CSC motifs of domains 1 (D1) and 3 (D3), respectively. The results on the wild-type protein confirmed earlier findings on the CCS-MBP (maltose binding protein) constructs, namely, that Cu(I) coordinates to the CXC motif, forming a cluster at the interface of two D3 polypeptides. In contrast to the single Cys to Ser mutations of the CCS-MBP protein (Stasser, J. P., Eisses, J. F., Barry, A. N., Kaplan, J. H., and Blackburn, N. J. (2005) Biochemistry 44, 3143-3152), single Cys to Ala mutations in D3 were sufficient to eliminate cluster formation and significantly reduce CCS activity. Analysis of the intensity of the Cu-Cu cluster interaction in C244A, C246A, and C244/246A variants suggested that the nuclearity of the cluster was greater than 2 and was most consistent with a Cu4S6 adamantane-type species. The relationship among cluster formation, oligomerization, and metal loading was evaluated. The results support a model in which Cu(I) binding converts the apo dimer with a D2-D2 interface to a new dimer connected by cluster formation at two D3 CSC motifs. The predominance of dimer over tetramer in the cluster-containing species strongly suggests that the D2 dimer interface remains open and available for sequestering an SOD1 monomer. This work implicates the copper cluster in the reactive form and adds detail to the cluster nuclearity and how copper loading affects the oligomerization states and reactivity of CCS for its partner SOD1.

Sco-like proteins contain copper bound by two cysteines and a histidine residue. Although their function is still incompletely understood, there is a clear involvement with the assembly of cytochrome oxidases which contain the Cu A center in subunit 2, possibly mediating the transfer of copper into the Cu A binuclear site. We are investigating the reaction chemistry of BSco, the homologue from B. subtilis. Our studies have revealed that BSco behaves more like a redox protein than a metallochaperone. The essential H135 residue which coordinates copper, plays a role in stabilizing the Cu(II) rather than the Cu(I) form. When H135 is mutated to alanine, the oxidation rate of both hydrogen peroxide and one-electron outer-sphere reductants increases by two orders of magnitude, suggestive of a redox switch mechanism between His-on and His-off conformational states of the protein. Imidazole binds to the H135A protein restoring the N superhyperfine coupling in the EPR, but is unable to rescue the redox properties of the WT Sco. These findings reveal a unique role for H135 in Sco function. We propose a hypothesis that electron transfer from Sco to the maturing oxidase may be essential for proper maturation and/or protection from oxidative damage during the assembly process. The findings also suggest that interaction of Sco with its protein partner(s) may perturb the Cu(II)-H135 interaction, and thus induce a sensitive redox activity to the protein.Sco1 is an essential accessory protein in the assembly of cytochrome-c-oxidase, the terminal enzyme of the respiratory chain. A large body of evidence links the function of Sco to the metalation of the Cu A center in subunit 2 of the oxidase (Cox2). In both yeast and B. subtilis, Sco mutant strains that impair or eliminate Cu binding produce a phenotype lacking in functional caa 3 oxidase, and high levels of Cu are able to rescue the caa 3 activity of B. subtilis-Sco (BSco) 1 -deficient strains (1-6). These data strongly implicate an interaction of † This work was supported in whole or in part by the National Institutes of Health Grant GM54803 (to NJB). This work was also supported 1 The abbreviations used are: BSco, Bacillus subtilis Sco; IPTG, isopropyl β-D-thiogalactopyranoside; WT, wild-type; NBT, nitroblue tetrazolium; BCIP, 5-bromo-4-chloro-3-indolylphosphate; TMPD, N, N, N′, N′-tetramethyl-p-phenylene diamine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EPR, electron paramagnetic resonance; EXAFS, extended X-ray absorption fine structure; XANES, X-ray absorption near edge structure. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 December 29. (14), and the Cu(II) form shows no tendency towards autoredox to disulfide and Cu(I), although this chemistry was observed in a crystal of the Ni(II) derivative (11). These considerations suggest that Sco-type proteins may exhibit two or more distinct activities which include both copper transfer and redox activities.To further understand the possible function of Sco ...

Sco is a red copper protein that plays an essential yet poorly understood role in the metalation of the Cu A center of cytochrome oxidase, and is stable in both the Cu(I) and Cu(II) forms. To determine which oxidation state is important for function, we constructed His135 to Met or selenomethionine (SeM) variants that were designed to stabilize the Cu(I) over the Cu(II) state. H135M was unable to complement a scoΔ strain of Bacillus subtilis, indicating that the His to Met substitution abrogated cytochrome oxidase maturation. The Cu(I) binding affinities of H135M and H135SeM were comparable to that of the WT and 100-fold tighter than that of the H135A variant. The coordination chemistry of the H135M and H135SeM variants was studied by UV/vis, EPR, and XAS spectroscopy in both the Cu(I) and the Cu(II) forms. Both oxidation states bound copper via the S atoms of C45, C49 and M135. In particular, EXAFS data collected at both the Cu and the Se edges of the H135SeM derivative provided unambiguous evidence for selenomethionine coordination. Whereas the coordination chemistry and copper binding affinity of the Cu(I) state closely resembled that of the WT protein, the Cu(II) state was unstable, undergoing autoreduction to Cu(I). H135M also reacted faster with H 2 O 2 than WT Sco. These data, when coupled with the complete elimination of function in the H135M variant, imply that the Cu(I) state cannot be the sole determinant of function; the Cu(II) state must be involved in function at some stage of the reaction cycle.

No abstract

No abstract