Copper Binding Site Research Articles

Reduction potential variations in azurin through secondary coordination sphere phenylalanine incorporations

Recent evidence has shown that the properties of metal binding sites can be tuned by more than the ligands in the primary coordination sphere. We investigated the incorporation of four phenylalanine residues into the secondary coordination sphere of the small soluble blue copper protein azurin. The locations for placement of these residues in azurin were based on the structure of the highly hydrophobic blue copper protein rusticyanin, which is known to have a significantly higher reduction potential than azurin. Using site-directed mutagenesis, these residues in close proximity to the copper binding site were mutated to large hydrophobic phenylalanine residues individually and in combination. We also added the Met121Leu mutation on top of the Phe mutations to construct a total of 13 variants. We found little change in the UV–visible absorption and EPR data for these proteins, however modest increases in reduction potential were observed with increases by as much as 30 mV per Phe residue. Furthermore, we observed the increases in potential to be additive.

Structure and Function of P19, a High-Affinity Iron Transporter of the Human Pathogen Campylobacter jejuni

Campylobacter jejuni, a major cause of acute bacterial diarrhea in humans, expresses numerous proteins to import diverse forms of essential iron. The expression of p19 and an adjacent iron transporter homologue ( ftr1) is strongly induced upon iron limitation, suggesting a function in iron acquisition. Here, we show that the loss of P19 alone is detrimental to growth on iron-restricted media. Furthermore, metal binding analysis demonstrates that recombinant P19 has distinct copper and iron binding sites. Crystal structures of P19 have been solved to 1.41 Å resolution, revealing an immunoglobulin-like fold. A P19 homodimer in which both monomers contribute ligands to two equivalent copper sites located adjacent to methionine-rich patches is observed. Copper coordination occurs via three histidine residues (His42, His95, and His132) and Met88. A solvent channel lined with conserved acidic residues leads to the copper site. Soaking crystals with a solution of manganese as iron analog reveals a second metal binding site in this solvent channel (metal–metal distance, 7.7 Å). Glu44 lies between the metal sites and displays multiple conformations in the crystal structures, suggesting a role in regulating metal–metal interaction. Dimerization is shown to be metal dependent in vitro and is detected in vivo by cross-linking.

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 A resolution using a twinned crystal.

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.

Disrupted zinc-binding sites in structures of pathogenic SOD1 variants D124V and H80R.

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we present structures of the pathogenic SOD1 variants D124V and H80R, both of which demonstrate compromised zinc-binding sites. The disruption of the zinc-binding sites in H80R SOD1 leads to conformational changes in loop elements, permitting non-native SOD1-SOD1 interactions that mediate the assembly of these proteins into higher-order filamentous arrays. Analytical ultracentrifugation sedimentation velocity experiments indicate that these SOD1 variants are more prone to monomerization than the wild-type enzyme. Although D124V and H80R SOD1 proteins appear to have fully functional copper-binding sites, inductively coupled plasma mass spectrometery (ICP-MS) and anomalous scattering X-ray diffraction analyses reveal that zinc (not copper) occupies the copper-binding sites in these variants. The absence of copper in these proteins, together with the results of covalent thiol modification experiments in yeast strains with and without the gene encoding the copper chaperone for SOD1 (CCS), suggests that CCS may not fully act on newly translated forms of these polypeptides. Overall, these findings lend support to the hypothesis that immature mutant SOD1 species contribute to toxicity in SOD1-linked ALS.

A joint x-ray and neutron study on amicyanin reveals the role of protein dynamics in electron transfer.

The joint x-ray/neutron diffraction model of the Type I copper protein, amicyanin from Paracoccus denitrificans was determined at 1.8 A resolution. The protein was crystallized using reagents prepared in D(2)O. About 86% of the amide hydrogen atoms are either partially or fully exchanged, which correlates well with the atomic depth of the amide nitrogen atom and the secondary structure type, but with notable exceptions. Each of the four residues that provide copper ligands is partially deuterated. The model reveals the dynamic nature of the protein, especially around the copper-binding site. A detailed analysis of the presence of deuterated water molecules near the exchange sites indicates that amide hydrogen exchange is primarily due to the flexibility of the protein. Analysis of the electron transfer path through the protein shows that residues in that region are highly dynamic, as judged by hydrogen/deuterium exchange. This could increase the rate of electron transfer by transiently shortening through-space jumps in pathways or by increasing the atomic packing density. Analysis of C-HX bonding reveals previously undefined roles of these relatively weak H bonds, which, when present in sufficient number can collectively influence the structure, redox, and electron transfer properties of amicyanin.

Spectroscopic and electrochemical studies of novel model compounds for cytochrome c oxidase

Different metalated porphyrin compounds were studied as model complexes for cytochrome c oxidase. All models contain a tyrosine molecule and a copper binding site. Two of the compounds are bearing an axial pyridine ligand that could possibly coordinate with Fe porphyrins. All complexes were studied using NMR and UV–Vis spectroscopies and it was found that the coordination of the axial ligand is possible only in one of the porphyrins. Moreover, the synthesized catalysts were studied as promising enzyme mimics using a rotating disc electrode in the presence of molecular oxygen.

Copper binds the carboxy-terminus of trefoil protein 1 (TFF1), favoring its homodimerization and motogenic activity.

Trefoil protein 1 (TFF1) is a small secreted protein belonging to the trefoil factor family of proteins, that are present mainly in the gastrointestinal (GI) tract and play pivotal roles as motogenic factors in epithelial restitution, cell motility, and other incompletely characterized biological processes. We previously reported the up-regulation of TFF1 gene in copper deficient rats and the unexpected property of the peptide to selectively bind copper. Following the previous evidence, here we report the characterization of the copper binding site by fluorescence quenching spectroscopy and mass spectrometric analyses. We demonstrate that Cys58 and at least three Glu surrounding residues surrounding it, are essential to efficiently bind copper. Moreover, copper binding promotes the TFF1 homodimerization, thus increasing its motogenic activity in in vitro wound healing assays. Copper levels could then modulate the TFF1 functions in the GI tract, as well as its postulated role in cancer progression and invasion.

Interactions between Copper-binding Sites Determine the Redox Status and Conformation of the Regulatory N-terminal Domain of ATP7B

Copper-transporting ATPase ATP7B is essential for human copper homeostasis and normal liver function. ATP7B has six N-terminal metal-binding domains (MBDs) that sense cytosolic copper levels and regulate ATP7B. The mechanism of copper sensing and signal integration from multiple MBDs is poorly understood. We show that MBDs communicate and that this communication determines the oxidation state and conformation of the entire N-terminal domain of ATP7B (N-ATP7B). Mutations of copper-coordinating Cys to Ala in any MBD (2, 3, 4, or 6) change the N-ATP7B conformation and have distinct functional consequences. Mutating MBD2 or MBD3 causes Cys oxidation in other MBDs and loss of copper binding. In contrast, mutation of MBD4 and MBD6 does not alter the redox status and function of other sites. Our results suggest that MBD2 and MBD3 work together to regulate access to other metal-binding sites, whereas MBD4 and MBD6 receive copper independently, downstream of MBD2 and MBD3. Unlike Ala substitutions, the Cys-to-Ser mutation in MBD2 preserves the conformation and reduced state of N-ATP7B, suggesting that hydrogen bonds contribute to interdomain communications. Tight coupling between MBDs suggests a mechanism by which small changes in individual sites (induced by copper binding or mutation) result in stabilization of distinct conformations of the entire N-ATP7B and altered exposure of sites for interactions with regulatory proteins.

Kinetic study of low density lipoprotein oxidation by copper

Oxidation of Low Density Lipoprotein (LDL) is regarded as a key event in the development of atherosclerosis. The aim of this study was to investigate effect of various copper concentrations on LDL oxidation kinetic profile as a mechanism in atherosclerosis process. LDL was isolated from plasma and its oxidation with copper was investigated by monitoring the formation of conjugated dienes. Based on time course of the formation of conjugated diene was observed at concentrations of 0.5 to 10 µM copper, represented the conventional kinetics of LDL oxidation with an inhibition period followed by a propagation phase. In contrast, at concentrations of 20 to 50 µM copper, LDL oxidation proceeded after a negligibly short lag-time followed by a distinct propagation phase. At lower copper concentrations of about 0.5 µM, LDL oxidation can be combined in 4 consecutive oxidation phase. The increasing copper concentration (to 10 µM) lowered the first propagation and shortened the seconded inhibition period until they melted into one apparent kinetic phase. But in copper concentrations of about 20 to 50 µM, increasing copper concentration increased the first propagation and the second inhibition but lowered the second propagation phase. The results of this investigation on the copper dependence of the oxidation kinetics suggest that LDL contains two different copper binding sites. Copper bound to the low affinity binding sites with molar ratio of 200 to 500 of copper / LDL. These ions bound to the high affinity binding sites with molar ratio of copper / LDL of 5 to 100.

Interactions of di-imine copper(II) complexes with albumin: competitive equilibria, promoted oxidative damage and DFT studies

Interactions of some diimine copper(II) complexes with bovine serum albumin (BSA) were investigated by spectroscopic techniques in order to compare the stability of the complexes and their capability of causing oxidative damage to the protein. The tri- and tetradentate imine ligands employed in this work contain pyridine, pyrazine or imidazole moieties, which are ubiquitous in biological systems. The relative thermodynamic stabilities of the copper(II) complexes were estimated by circular dichroism (CD) using BSA as the competitive ligand. The apparent stability constants determined for the complexes are very similar to one another and to that of the Cu(BSA) complex itself, for which log K Cu(BSA) = 12.9 has already been described in the literature, indicating that the complexes are quite stable under physiological conditions. Two different copper binding sites were evidenced on BSA by spectroscopic measurements (CD, UV-Vis and EPR), depending on the ligand and on the [CuL]:[protein] stoichiometric ratio. Metal binding to any of the sites gives rise to significant protein oxidative damage, especially in the presence of hydrogen peroxide, indicating an oxidative process based on reactive oxygen species (ROS). A small amidated peptide, Asp-Thr-His-NH2, corresponding to the N-terminal region of BSA was synthesized, and its interaction with all the diimine-copper(II) complexes was also investigated in order to clarify the copper imine complex-albumin interactions. Electronic structure calculations at the density functional theory (DFT) level were made to compare the copper-ligand binding energies for each complex with that of the metal coordinated at the N-terminal site of the protein.

Characterization of Partial Reactions in the Catalytic Cycles of Calcium and Copper ATPases

Calcium and copper P-type ATPases transduce ATP chemical energy to transmembrane osmotic energy, using a catalytic mechanism common to haloacid dehalogenases where phosphoryl transfer from substrate to a conserved aspartate yields a phosphoenzyme intermediate before hydrolytic cleavage of Pi. Favored by its native abundance, characterization of the calcium ATPase is by now quite detailed, including two transmembrane calcium binding sites, a phosphorylation site within the headpiece, a conserved TGES motif for catalytic assistance of hydrolytic cleavage, and domain movements permitting long range linkage of phosphorylation and calcium binding sites. Presently, mutational analysis is still yielding further details on the calcium ATPase. As for bacterial (CopA) and human (ATP7B) copper ATPases, due to low native abundance, heterologous expression in cultured cells is required. In addition to domains present in other P-type ATPases, copper ATPase sequences include an amino terminus extension (NMBD) with one (CopA) or six (ATP7B) copper binding sites. Using recombinant protein, it is possible to demonstrate formation of phosphoenzyme intermediate by utilization of ATP, undergoing rather slow turnover. In analogy to the calcium ATPase, phosphoenzyme intermediate is not formed following mutation of the conserved aspartate or the transmembrane copper site. In addition to the phosphorylated ATPase intermediate, copper dependent phosphorylation of various serine residues occurs in ATP7B. Interference with protein autophosphorylation, both of aspartate and serines, is observed following mutation of a histidine residue in the nucleotide binding domain, a mutation found in Wilson disease of humans. The effects of NMBD copper site mutations and deletions suggest that the NMBD sequence is involved in catalytic regulation as well as protein targeting. (Supported by 5 R01 HL069830-08).

Modelling Copper Binding to the Amyloid-β Peptide in Alzheimer

Oxidative modification due to reactive oxygen species generated by Cu2+ bound to the amyloid-β peptide may be one of the sources of neurodegeneration observed in Alzheimer’s disease. Understanding the structure and function of the copper binding site can assist in the design of effective therapeutics. This paper highlights some of the most significant recent developments in computational modelling studies of the structure of the binding site and reaction mechanisms of reactive oxygen species generation.

Cloning, expression in Escherichia coli, and enzymatic properties of laccase from Aeromonas hydrophila WL-11

A strain WL-11 with high laccase activity was isolated from activated sludge collected from the effluent treatment plant of a textile and dyeing industry. It was identified as Aeromonas hydrophila by physiological test and 16S rDNA sequence analysis. A gene encoding of laccase from a newly isolated Aeromonas hydrophila WL-11 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 1605 bp encoding a polypeptide comprised of 534 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other laccases, including the conserved regions of four histidine-rich copper-binding sites. The predicted amino acid sequence showed a high homology (more than 60%) with bacterial laccases in the genome and protein databases and the highest degree of similarity (61% identity) was observed with the multicopper oxidase of Klebsiella sp. 601. When expressed in Escherichia coli, the recombinant enzyme was overproduced in the cytoplasm as soluble and active form. The purified enzyme had an optimum pH of 2.6 and 8.0 for ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid) and DMP (2,6-dimethoxyphenol), respectively. The kinetic study on ABTS revealed a higher affinity of this enzyme to this substrate than DMP.

Modeling Cu(II) Binding to Peptides Using the Extensible Systematic Force Field

The utility of the extensible systematic force field (ESFF) was tested for copper(II) binding to a 34-amino-acid Cu(II) peptide, which includes five histidine residues and is the putative copper-binding site of lysyl oxidase. To improve computational efficiency, distance geometry calculations were used to constrain all combinations of three histidine ligands to be within bonding distance of the copper and the best results were utilized as starting structures for the ESFF computations. All likely copper geometries were modeled, but the results showed only a small dependence on the geometrical model in that all resulted in a distorted square pyramidal geometry about the copper, some of the imidazole rings were poorly oriented for ligation to the Cu(II), and the copper-nitrogen bond distances were too long. The results suggest that ESFF should be used with caution for Cu(II) complexes where the copper-ligand bonds have significant covalency and when the ligands are not geometrically constrained to be planar.

Tyrosinase gene (TYR) mutations in Chinese patients with oculocutaneous albinism type 1

To identify the sequence variants of the tyrosinase (TYR) gene in Chinese families with oculocutaneous albinism. Three families with oculocutaneous albinism type 1 and 95 unrelated healthy Chinese individuals with normal pigmentation were screened for mutations in the TYR gene by direct sequencing. Computational algorithms were used to characterize the biological significance of the mutants. Four previously reported mutations (R299C, R299H, W400L and frame-shift c.930insC) and one novel mutation (F214del) were identified, and probands had homozygous or compound heterozygous TYR mutant alleles. None of the mutants were identified among the 95 normal control subjects. Computational analysis predicted that the R299C mutant inactivates the tyrosinase enzyme by misfolding of protein tertiary structure and/or retention of the misfolded tyrosinase within the endoplasmic reticulum, and F214del causes dysfunction of tyrosine enzyme by affecting the copper binding sites and altering substrate orientation and electronic transfers during catalytic reactions for melanosynthesis. We have identified five different TYR mutations, including one novel mutation, which caused oculocutaneous albinism type 1 in Chinese. Further analysis of the patients will be useful to determine the effects of these mutations on the tyrosinase activities.

Crystal structure of human copper homeostasis protein CutC reveals a potential copper-binding site

Copper is an essential trace element to life and particularly plays a pivotal role in the physiology of aerobic organisms. The Cut protein family is associated with copper homeostasis and involved in uptake, storage, delivery, and efflux of copper. CutC is a member of the Cut family and is suggested to be involved in efflux trafficking of cuprous ion. We report here the biochemical and structural characterization of human CutC (hCutC). hCutC can bind Cu(I) with a stoichiometry of 1:1 and an apparent dissociation constant of 15.5+/-2.8 microM. hCutC assumes a typical TIM-barrel fold and forms a tetramer in both crystal structure and solution which is different from the dimeric architecture of the bacterial CutC. Structure analysis and sequence comparison of CutC proteins from different species reveal two strictly conserved Cys residues on the inner surface of the C-terminal end of the TIM-barrel. Mutations of the two Cys residues can significantly impair the binding ability of hCutC with Cu(I). Our results suggest that hCutC functions as an enzyme with Cu(I) as a cofactor rather than a copper transporter and the potential Cu(I)-binding site consists of the two Cys residues and other conserved residues in the vicinity.

The Second Cu(II)-Binding Site in a Proton-Rich Environment Interferes with the Aggregation of Amyloid-β(1−40) into Amyloid Fibrils

The overall morphology and Cu(II) ion coordination for the aggregated amyloid-beta(1-40) [Abeta(1-40)] in N-ethylmorpholine (NEM) buffer are affected by Cu(II) ion concentration. This effect is investigated by transmission electron microscopy (TEM), atomic force microscopy (AFM), and electron spin echo envelope modulation (ESEEM) spectroscopy. At lower than equimolar concentrations of Cu(II) ions, fibrillar aggregates of Abeta(1-40) are observed. At these concentrations of Cu(II), the monomeric and fibrillar Abeta(1-40) ESEEM data indicate that the Cu(II) ion is coordinated by histidine residues. For aggregated Abeta(1-40) at a Cu(II):Abeta molar ratio of 2:1, TEM and AFM images show both linear fibrils and granular amorphous aggregates. The ESEEM spectra show that the multi-histidine coordination for Cu(II) ion partially breaks up and becomes exposed to water or exchangeable protons of the peptide at a higher Cu(II) concentration. Since the continuous-wave electron spin resonance results also suggest two copper-binding sites in Abeta(1-40), the proton ESEEM peak may arise from the second copper-binding site, which may be significantly involved in the formation of granular amorphous aggregates. Thioflavin T fluorescence and circular dichroism experiments also show that Cu(II) inhibits the formation of fibrils and induces a nonfibrillar beta-sheet conformation. Therefore, we propose that Abeta(1-40) has a second copper-binding site in a proton-rich environment and the second binding Cu(II) ion interferes with a conformational transition into amyloid fibrils, inducing the formation of granular amorphous aggregates.

Copper binding to chicken and human prion protein amylodogenic regions: Differences and similarities revealed by Ni2+ as a diamagnetic probe

Both human (h) and chicken (Ch) prion proteins (PrP) bind copper ions within the so called "tandem repeat" N-terminal region. Outside this region, hPrP possesses two additional copper binding sites, localized at His-96 and His-111 in the so called "amylodogenic" or neurotoxic region (residues 91-126). Also ChPrP possesses a similar region (ChPrP(105-140)) containing two His (His-110 and His-124) and an identical hydrophobic tail of 15 amino acids rich in Ala and Gly. The copper binding abilities within such region of ChPrP were investigated by NMR, CD and potentiometry using Ni(2+) as diamagnetic probe. The formation of diamagnetic metal complexes allowed to monitor the chemical shift and signal intensity variations and to determine the structural and kinetic features of the His-110 and His-124 metal binding sites. Finally a comparison between the hPrP and ChPrP metal binding abilities was performed. We found that the two prion proteins exhibited different copper and nickel preferences with the favoured metal binding sites localized at opposite His: His-110 for ChPrP, and His-111 for hPrP.

Site-directed mutagenesis reveals a conservation of the copper-binding site and the crucial role of His24 in CopH from Cupriavidus metallidurans CH34

CopH is a periplasmic copper-binding protein from Cupriavidus metallidurans CH34 that contains two histidine residues. Both His24 and His26 contribute to the formation of two high-affinity copper-binding sites in wild-type CopH and are likely involved in a 2N2O coordination sphere in the equatorial plane. We have used site-directed mutagenesis, and a series of spectroscopic and calorimetric studies to further characterize the copper-binding sites in CopH. While His24 plays a predominant role in copper affinity, one Cu-binding site was lost when either histidine residue was mutated. However, as shown by NMR and EPR, the mutation of the His residues does not affect the structural organization of the Cu-binding site nor the number of nitrogen ligands involved in copper ligation. In the absence of structural data, we propose a model that conciliates most of the spectroscopic data recorded during this study.

High Yield Heterologous Expression of Wild-type and Mutant Cu+-ATPase (ATP7B, Wilson Disease Protein) for Functional Characterization of Catalytic Activity and Serine Residues Undergoing Copper-dependent Phosphorylation

ATP7B is a P-type ATPase required for copper homeostasis and related to Wilson disease of humans. In addition to various domains corresponding to other P-type ATPases, ATP7B includes an N terminus extension (NMBD) with six copper binding sites. We obtained high yield expression of WT and mutant ATP7B in COS1 cells infected with adenovirus vector. ATP7B, isolated with the microsomal fraction of cell homogenates, accounts for 10–20% of the total protein. Copper-dependent, steady-state ATPase yields 30 nmol of Pi/mg of protein/min at 37 °C, pH 6.0. ATP7B phosphorylation with ATP occurs with diphasic kinetics and is totally copper-dependent. Alkali labile phosphoenzyme (catalytic intermediate of P-ATPases) accounts for a small fraction of the total phosphoprotein and is prevented by D1027N (P domain) or C983A/C985A (CXC copper binding motif in TM6) mutations. Decay of [32P]phosphoenzyme following chase with non-radioactive ATP occurs with an initial burst involving alkali labile phosphoenzyme (absent in D1027N and C983A/C985A mutants) and continues at a slow rate involving alkali-resistant phosphoenzyme. If a copper chelator is added with the ATP chase, the initial burst is smaller, and further cleavage is totally inhibited. Analysis by proteolysis and mass spectrometry demonstrates that the alkali stable phosphoenzyme involves Ser478 and Ser481 (NMBD), Ser1121 (“N” domain) and Ser1453 (C terminus), and occurs with the same pattern ex vivo (COS-1) and in vitro (microsomes). The overall copper dependence of phosphorylation and hydrolytic cleavage suggests long range conformational effects, including interactions of NMBD and headpiece domains, with strong influence on catalytic turnover.