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.