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Link to Free Full Text: ............................................................................................................................................ https://onlinelibrary.wiley.com/share/author/J5YPUTRMDWJNMENJYC8K?target=10.1002/cbdv.202100043 ............................................................................................................................................ Proteins anchor copper(II) ions mainly by imidazole from histidine residues located in different positions in the primary protein structures. However, the motifs with histidine in the first three N-terminal positions (His1, His2, and His3) show unique Cu(II)-binding properties, such as availability from the surface of the protein, high flexibility, and high Cu(II) exchangeability with other ligands. It makes such sequences beneficial for the fast exchange of Cu(II) between ligands. Furthermore, sequences with His1 and His2, thus, non-saturating the Cu(II) coordination sphere, are redox-active and may play a role in Cu(II) reduction to Cu(I). All human protein sequences deposited in UniProt Knowledgebase were browsed for those containing His1, His2, or His3. Proteolytically modified sequences (with the removal of a propeptide or Met residue) were taken for the analysis. Finally, the sequences were sorted out according to the subcellular localization of the proteins to match the respective sequences with the probability of interaction with divalent copper.
Chem. Biodiversity 2021; 18: e2100043
Cu(II)-binding N-terminal sequences of human proteins
Tomasz Frączyk
tfraczyk@ibb.waw.pl
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... In organisms, copper ions mainly exist in the form of complexes with proteins or peptides, although small molecule-based metal chelators can be used to maintain the concentration of free copper ions below picomolar levels. In peptide-copper complexes, the terminal amino group, deprotonated amide nitrogen and various side chain donors are the binding sites of copper ions [3][4][5][6][7][8][9]. Histidine residues in the peptide sequence, especially in the first three positions of the N-terminal (denoted as His1, His2, and His3), are the main sites for the binding of copper ions. ...
... Histidine residues in the peptide sequence, especially in the first three positions of the N-terminal (denoted as His1, His2, and His3), are the main sites for the binding of copper ions. For example, the His1-contained peptide can bind Cu 2+ by two nitrogen atoms in the free amine of the N-terminal and the imidazole group of histidine residue [3,9], the His2-contained peptide can bind Cu 2+ by three nitrogen atoms in the free amine of N-terminal, the second amide as well as the imidazole group of histidine residue [6], and the His3-contained peptide, also denoted as the amino-terminal copper and nickel (ATCUN) binding peptide, can chelate Cu 2+ and Ni 2+ ions with femtomolar affinity. Cu 2+ is coordinated by ATCUN peptide through the N-terminal free amine, the second and third amides, and the imidazole group of histidine residue [6][7][8][9]. ...
... For example, the His1-contained peptide can bind Cu 2+ by two nitrogen atoms in the free amine of the N-terminal and the imidazole group of histidine residue [3,9], the His2-contained peptide can bind Cu 2+ by three nitrogen atoms in the free amine of N-terminal, the second amide as well as the imidazole group of histidine residue [6], and the His3-contained peptide, also denoted as the amino-terminal copper and nickel (ATCUN) binding peptide, can chelate Cu 2+ and Ni 2+ ions with femtomolar affinity. Cu 2+ is coordinated by ATCUN peptide through the N-terminal free amine, the second and third amides, and the imidazole group of histidine residue [6][7][8][9]. In general, the location of histidine in the peptide sequence has a decisive impact on the characteristics of peptide-copper complexes, including the coordination mode, binding affinity, redox potential, and catalytic activity. ...
Article
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Copper ions, as the active centers of natural enzymes, play an important role in many physiological processes. Copper ion-based catalysts which mimic the activity of enzymes have been widely used in the field of industrial catalysis and sensing devices. As an important class of small biological molecules, peptides have the advantages of easy synthesis, excellent biocompatibility, low toxicity, and good water solubility. The peptide–copper complexes exhibit the characteristics of low molecular weight, high tenability, and unique catalytic and photophysical properties. Biosensors with peptide–copper complexes as the signal probes have promising application prospects in environmental monitoring and biomedical analysis and diagnosis. In this review, we discussed the design and application of fluorescent, colorimetric and electrochemical biosensors based on the peptide–copper coordination interaction.
... The copper ion-anchoring proteins always contain histidine (His) residues located in different positions in the primary sequence. It has been demonstrated that the number and position of His residues have a significant influence on the coordination modes of copper ion-protein complexes [16][17][18][19]. Especially, the location of His residue in the first three Nterminal positions (His 1 , His 2 , and His 3 ) can alter the metal binding affinity of the peptide chain and the catalytic activities of copper [17,20,21]. ...
... It has been demonstrated that the number and position of His residues have a significant influence on the coordination modes of copper ion-protein complexes [16][17][18][19]. Especially, the location of His residue in the first three Nterminal positions (His 1 , His 2 , and His 3 ) can alter the metal binding affinity of the peptide chain and the catalytic activities of copper [17,20,21]. For this consideration, the aminoterminal Cu and Ni-binding (ATCUN) motif with the general sequence of H 2 N-Xxx-Zzz-His (XZH) at the N-terminus can be introduced into proteins and peptides for anticancer or antimicrobial applications [20,[22][23][24]. ...
... Histidine residue in the peptide chain plays a decisive role in the binding of copper ions. For example, the peptide with a His 1 motif in the N-terminal can coordinate with Cu(II) in a non-saturating binding format [16,17]. Our group found that the peptide with a His 3 motif in the N-terminal position (known as ATCUN) shows a unique Cu(II)-binding property and redox activity. ...
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Proteases play a critical role in regulating various physiological processes from protein digestion to wound healing. Monitoring the activity of proteases and screening their inhibitors as potential drug molecules are of great importance for the early diagnosis and treatment of many diseases. In this work, we reported a general, label-free and homogeneous electrochemical method for monitoring protease activity based on the peptide–copper interaction. Cleavage of peptide substrate results in the generation of a copper-binding chelator peptide with a histidine residue in the first or third position (His1 or His3) at the N-terminal. The redox potential and current of copper coordinated with the product are different from the free copper or the copper complex with the substrate, thus allowing for the detection of protease activity. Angiotensin-converting enzyme (ACE) and thrombin were determined as the model analytes. The label-free and homogeneous electrochemical method can be used for screening protease inhibitors with high simplicity and sensitivity.
... Because phosphate anions may influence the Cu(II)-peptide species, the latter buffer better reflects the conditions in the human body. [12,25] Here, the redox behaviour of Cu(II) complexes with Aβ(11-16) (peptide sequence: EVHHQKam) and its pyroglutamate counterpart pAβ(11-16) (sequence pEVHHQK-am) was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in two kinds of solutions: 4 mM HNO3/96 mM KNO3 and 0.1 M phosphate buffer. Aβ (11)(12)(13)(14)(15)(16) belongs to the ATCUN peptide family, and therefore, the Cu(II) ion is bonded via four nitrogens by the EVH sequence (creating 4N complexes) in a square-planar geometry. ...
... 4N structures. Despite this, Fraczyk, using spectroscopic techniques, showed that phosphorylation of serine residues markedly weakens the stability of ATCUN-Cu(II) complexes.[25] Up until now, there are few voltammetric studies where the influence of phosphoric ions on ATCUN complex properties are investigated. ...
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Here we demonstrate a significant difference in redox behaviour of copper complexes with β-amyloids Aβ(11-x) and pAβ(11-x) which are models for important components of senile plaques. A small change in the peptide chain may enhance reactive oxygen species (ROS) formation which can severely damage nerve cells.
... [9] The ATCUN motif (amino terminal Cu(II) and Ni(II) binding) occurs naturally at the Nterminus of albumins, [10] the Cu transport protein Ctr1, [11] neuromedin C [12] and several other (neuro)hormones and immune system-related peptides. [13] Under physiological conditions it binds Cu(II) in a square planar fashion (4N chelating ligand) through the amine of the N-terminal amino acid, two deprotonated amide bonds and the δ-N atom (Nim) of the imidazole moiety of His in position 3 ( Figure 1). [10][11][12][13][14] In 1983, Pauling et al. showed that the Cu(II) complex with the simplest ATCUN motif, the tripeptide NH2-Gly-Gly-His-COOH (Cu-GGH), killed Ehrlich ascites tumor cells in vivo, and cleaved DNA in the presence of the reducing agent ascorbate (ascH -) and O2. ...
... [13] Under physiological conditions it binds Cu(II) in a square planar fashion (4N chelating ligand) through the amine of the N-terminal amino acid, two deprotonated amide bonds and the δ-N atom (Nim) of the imidazole moiety of His in position 3 ( Figure 1). [10][11][12][13][14] In 1983, Pauling et al. showed that the Cu(II) complex with the simplest ATCUN motif, the tripeptide NH2-Gly-Gly-His-COOH (Cu-GGH), killed Ehrlich ascites tumor cells in vivo, and cleaved DNA in the presence of the reducing agent ascorbate (ascH -) and O2. [15] Cowan et al. reported Cu(II) ATCUN complexes with anticancer, -viral, and -microbial activity as well as for DNA cleavage and enzyme inhibition. ...
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Redox active Cu(II) complexes are able to form reactive oxygen species (ROS) in the presence of oxygen and reducing agents. Recently, Faller et al. reported that ROS generation by Cu(II) ATCUN complexes is not as high as assumed for decades. High complex stability results in silencing of the Cu(II)/Cu(I) redox cycle and therefore to low ROS generation. In this work, we demonstrate that an exchange of the a ‐amino acid Gly with the β‐amino acid β‐Ala at position 2 (Gly2 → β‐Ala2) of the ATCUN motif reinstates ROS production ( . OH and H 2 O 2 ). Potentiometry, cyclic voltammetry, EPR spectroscopy and DFT simulations were utilized to explain the increased ROS generation of these β‐Ala2‐containing ATCUN complexes. We also observed enhanced oxidative cleavage activity towards plasmid DNA for β‐Ala2 compared to the Gly2 complexes. Modifications with positively charged Lys residues increased the DNA affinity through electrostatic interactions as determined by UV/VIS, fluorescence, and CD spectroscopy, and consequently led to a further increase in nuclease activity. A similar trend was observed regarding the cytotoxic activity of the complexes against several human cancer cell lines where β‐Ala2 peptide complexes had lower IC 50 values compared to Gly2. The higher cytotoxicity could be attributed to an increased cellular uptake as determined by ICP‐MS measurements.
... For a great majority of copper ion-containing proteins, histidine (His) residue located in different positions of peptide sequence has an important effect on the coordination of copper ion [18]. For example, peptides with a His residue in the first, second, or third position of N-terminal show different affinity capability and coordination mode towards copper ion [19][20][21][22][23][24]. We have reported that the peptide with a histidine (His) residue in the third position of N-terminal (denoted as ATCUN peptide) can limit the peroxidase and oxidase-like catalytic ability of copper ion, but the ATCUN peptide-copper complexes exhibit excellent activity for electrocatalytic water oxidation [25][26][27]. ...
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This work suggested that Cu2+ ion coordinated by the peptide with a histidine (His or H) residue in the first position from the free N-terminal reveals oxidase-mimicking activity. A biotinylated polymer was prepared by modifying His residues on the side chain amino groups of lysine residues (denoted as KH) to chelate multiple Cu2+ ions. The resulting biotin-poly-(KH-Cu)20 polymer with multiple catalytic sites was employed as the signal label for immunoassay. Prostate specific antigen (PSA) was determined as the model target. The captured biotin-poly-(KH-Cu)20 polymer could catalyze the oxidation of o-phenylenediamine (OPD) to produce fluorescent 2,3-diaminophenazine (OPDox). The signal was proportional to PSA concentration from 0.01 to 2 ng/mL, and the detection limit was found to be eight pg/mL. The high sensitivity of the method enabled the assays of PSA in real serum samples. The work should be valuable for the design of novel biosensors for clinical diagnosis.
... 76 This idea is further supported by the ability of ternary ligands, both imidazoles and thiols, to tune its rate. Analogous species could also form and act intracellularly, given the abundance of XH motifs in cellular proteins, 77 and the known strict homology of various XH motifs in terms of Cu(II) coordination properties. 49−52 ...
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Gly-His-Lys (GHK) is a tripeptide present in the human bloodstream that exhibits a number of biological functions. Its activity is attributed to the copper-complexed form, Cu(II)GHK. Little is known, however, about the molecular aspects of the mechanism of its action. Here, we examined the reaction of Cu(II)GHK with reduced glutathione (GSH), which is the strongest reductant naturally occurring in human plasma. Spectroscopic techniques (UV–vis, CD, EPR, and NMR) and cyclic voltammetry helped unravel the reaction mechanism. The impact of temperature, GSH concentration, oxygen access, and the presence of ternary ligands on the reaction were explored. The transient GSH-Cu(II)GHK complex was found to be an important reaction intermediate. The kinetic and redox properties of this complex, including tuning of the reduction rate by ternary ligands, suggest that it may provide a missing link in copper trafficking as a precursor of Cu(I) ions, for example, for their acquisition by the CTR1 cellular copper transporter.
... 11 A recent study indicated that more peptides with such properties remain to be identified in human proteome. 12 Peptides have also been used extensively to model Cu(II) binding to its transport proteins, such as albumin and hCtr1 membrane transporter, 13−15 and synaptic proteins, such as prions, APP, and αsynuclein. 16,17 Two N-terminal sequence motifs, Xaa-His and Xaa-Zaa-His (where Xaa is any α-amino acid except of Cys, and Zaa is any α-amino acid except of Cys or Pro), provide the highest Cu(II) complex affinities by virtue of synergistic formation of chelate rings involving peptide nitrogen atoms ( Figure 1). ...
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The toolset of mass spectrometry (MS) is still expanding, and the number of metal ion complexes researched this way is growing. The Cu(II) ion forms particularly strong peptide complexes of biological interest which are frequent objects of MS studies, but quantitative aspects of some reported results are at odds with those of experiments performed in solution. Cu(II) complexes are usually characterized by fast ligand exchange rates, despite their high affinity, and we speculated that such kinetic lability could be responsible for the observed discrepancies. In order to resolve this issue, we selected peptides belonging to the ATCUN family characterized with high and thoroughly determined Cu(II) binding constants and re-estimated them using two ESI-MS techniques: standard conditions in combination with serial dilution experiments and very mild conditions for competition experiments. The sample acidification, which accompanies the electrospray formation, was simulated with the pH–jump stopped-flow technique. Our results indicate that ESI-MS should not be used for quantitative studies of Cu(II)–peptide complexes because the electrospray formation process compromises the entropic contribution to the complex stability, yielding underestimations of complex stability constants.
... 1,2 It specifically binds Cu 2+ via an amino-terminal H 2 N− Xaa−Yaa−His sequence (Figure 1a), 3,4 which is found in more than 100 human extracellular proteins and peptides. 5 Serum contains numerous low-molecular-weight ligands, 6−8 including the tripeptide glycyl-L-histidyl-L-lysine (GHK), a growth factor found at its highest concentration in the albumin-rich fraction of plasma 9−11 and proposed to exert biological actions in its Cu 2+ -bound form. 12,13 Also found in saliva and urine, it is thought to be released into the bloodstream by proteolysis of extracellular proteins osteonectin and type I collagen. ...
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