Active-Site Environment of Copper-Bound Human ... - ACS Publications

Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

pubs.acs.org/IC

Active-Site Environment of Copper-Bound Human Amylin Relevant to Type 2 Diabetes Manas Seal and Somdatta Ghosh Dey* Department of Inorganic Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India S Supporting Information *

ABSTRACT: Type 2 diabetes mellitus (T2Dm) is characterized by reduced β cell mass and amyloid deposits of human islet amyloid polypeptide (hIAPP) or amylin, a 37 amino acid containing peptide around pancreatic β cells. The interaction of copper (Cu) with amylin and its mutants has been studied in detail using absorption, circular dichroism, electron paramagnetic resonance spectroscopy, and cyclic voltammetry. Cu binds amylin in a 1:1 ratio, and the binding domain lies within the first 19 amino acid residues of the peptide. Depending on the pH of the medium, Cu-amylin shows the formation of five pH-dependent components (component IV at pH 4.0, component III at pH 5.0, component II at pH 6.0, component I at pH 8.0, and another higher pH component above pH 9.0). The terminal amine, His18, and amidates are established as key residues in the peptide that coordinate the Cu center. The physiologically relevant components I and II can generate H2O2, which can possibly account for the enhanced toxicity of amylin in the presence of Cu, causing damage of the β cells of the pancreas via oxidative stress. diseases.13,14 Metal ions are also critically involved in the pathology of T2Dm.15 Zinc (Zn), iron (Fe), and copper (Cu) have been associated with T2Dm.16−18 The concentration of Zn in pancreatic β cells is very high, and Zn deficiency is common in T2Dm.16 An increased dietary intake of Fe, especially in the form of heme, enhances the risk of T2Dm.19−21 Heme can bind amylin to form a heme−amylin complex, which can contribute to oxidative stress via the formation of hydrogen peroxide (H2O2).22,23 On the other hand, significantly higher levels of Cu are found in the serum of diabetic patients.24 It has been observed that Cu can increase amylin-induced apoptosis from 45% to 70% in cultured β cells.25 On the other hand, in the presence of Cu chelating agents, no enhanced toxicity was observed.25 Previous studies suggest that Cu can interact with amylin and delay aggregation.26,27 It has been proposed that Cu binding to monomeric amylin competes with the conformation required to form aggregate and thus enhances the oligomeric form of amylin which is relatively more toxic.25,28,29 Moreover, in the reduced state, Cu can produce partially reduced oxygen species (PROS) like superoxide, peroxide, etc., which can cause severe damage to cells and cellular components.25,30 Amylin itself increases H2O2 levels when exposed to a cultured β cell, and the formation of H2O2 is enhanced in manifolds by the presence of Cu.25,31,32 Interestingly, pancreatic β cells are extremely prone to oxidative damage because of low expression

1. INTRODUCTION Diabetes mellitus (Dm) is a chronic disorder mainly characterized by an impaired glucose metabolism arising from defects in insulin activity and its deficiency in the pancreatic β cells.1 More than 400 million people are suffering from diabetes worldwide and 90% of the patients suffer from type 2 diabetes mellitus (T2Dm).2 The increasing number of patients with T2Dm is an alarming situation to future world. Like Alzheimer’s and Parkinson’s disease, T2Dm also follows a common pathological mechanism of protein aggregation.3,4 The pathological feature of T2Dm is the presence of amyloid deposits around pancreatic β cells.5,6 These deposits are known to be composed of a disordered hormone called amylin or human islet amyloid polypeptide (hIAPP), which is coproduced with insulin, is trafficked through the similar secretory pathway of insulin, and gets deposited around the β cells.7 Amylin is a hydrophobic polypeptide, constituted of 37 amino acids.8 It contains a disulfide bond between Cys2 and Cys7 and is amidated at the C-terminus. Several reports indicate that oligomeric forms of amylin disrupt cell membranes and cause apoptosis.9 The pathway of β cell death involves phenomena like cell membrane blebbing, chromatin condensation, mitochondrial dysfunction, DNA fragmentation, and oxidative stress.10,11 Adverse effects of metal ions have been observed in diseases like Alzheimer’s, Parkinson’s, Wilson, Menkes, Prion, and Huntington, which develop via protein aggregation.12,13 Metal-ion coordination to the peptides, which are hallmarks of the diseases, may explain the molecular basis for the macroscopic development of the © XXXX American Chemical Society

Received: September 4, 2017

A

DOI: 10.1021/acs.inorgchem.7b02266 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

Scheme 1. Amino Acid Sequences of (A) Human Amylin(1−37) Showing the Hydrophilic Region in Blue, (B) Human Amylin(1−19), (C) Human Amylin(1−17), (D) N-Terminal Acylated Human Amylin(1−19), NAc-Amylin(1−19), (E) 2,7-Gly Mutant of Human Amylin(1−19), 2,7-Gly-Amylin(1−19), and (F) Rat Amylin(1−37), Highlighting the Differences with Human Amylin(1−37)a

a

All of the peptides are C-terminally amidated and contain disulfide linkages between Cys at the 2nd and 7th positions. 2,7-Gly-amylin(1−19) does not contain any disulfide linkages.

of antioxidant enzymes like catalase or glutathione peroxidase.33 The increased production of reactive oxygen species decreases insulin gene expression and its secretion, which finally leads to β cell apoptosis.34−36 Hence, interaction of Cu with amylin, enhanced toxicity due to PROS formation, and increasing oligomeric form of amylin are critical factors for the development of T2Dm. Previously, the interaction of Cu with amylin has been described by several groups.26,37−40 A thorough investigation of the active-site environment of a Cuamylin complex would help to understand the critical role of Cu in T2Dm. The present study involves the interaction of Cu with native amylin(1−37) as well as the truncated hydrophilic region of the peptides and characterization of the active-site environment of Cu-bound amylin and its site directed mutants using absorption, circular dichroism, electron paramagnetic resonance spectroscopy, and cyclic voltammetry. The coordination environment of Cu-amylin at various pH values has been elucidated. The reduced Cu-bound amylin is found to generate a substantial amount of H2O2, which possibly accounts for the oxidative stress associated with T2Dm.

measured with a micro pH meter before each experiment. To measure the amount of reduced oxygen species by a reduced Cu-amylin complex, all of the experiments were performed in a degassed buffer prepared via a freeze−pump−thaw cycle. 2.3. Absorption and Circular Dichroism (CD) Spectroscopy. All of the absorption data were recorded by a UV−vis diode-array spectrophotometer (Agilent 8453). CD spectra were obtained by a Jasco (J-815) CD spectrometer. For both absorption and CD, cuvettes of 1 cm path length were used. The absorption and CD data were simultaneously fitted using the program PeakFit 4.0. Minimum numbers of bands were used to adequately fit key features in both spectra. 2.4. Electron Paramagentic Resonance (EPR) Spectroscopy. EPR spectra were obtained by a JEOL (JES FA200) spectrophotometer. EPR samples were 0.5 mM in concentration and were run at 77 K in a liquid-nitrogen finger Dewar. The microwave power was 1 mW, the center field was 320 mT, the time constant was 0.03, and the modulation width was 1.6 mT. The pH perturbation study in EPR was performed with the same solution, and the pH was checked before and after the EPR spectrophotometer was run. 2.5. Cyclic Voltammetry (CV). CV was performed on a CH Instruments potentiostat (model 710D). The concentrations of the Cu-amylin complexes were 0.1−0.2 mM. Pt, Ag/AgCl, and glassy carbon were used as counter, reference, and working electrodes, respectively. 2.6. Partially Reduced Oxygen Species (PROS) Detection. For PROS calculation, a xylenol orange assay was performed as follows. A total of 4.9 mg of Mohr’s salt and 3.9 mg of xylenol orange were dissolved in 5 mL of 250 mM H2SO4, and the resulting solution was stirred for 10 min. A total of 200 μL of this solution was taken in 1.8 mL of nanopure water, and a calibration curve for the quantitative estimation of H2O2 was obtained for 0.05, 0.1, 0.5, 1, 2.5, 5, and 10 μM concentrations of H2O2 by recording their absorbance at 560 nm (Figure S18). The calibration curve was expressed as the absorbance at a fixed wavelength of 560 nm versus concentration of H2O2 in micromolar units for a 2 mL volume. For the detection of PROS of an unknown quantity, a blank was obtained in the UV−vis spectrophotometer with 1.8 mL of nanopure water in a cuvette. A total of 200 μL of the xylenol orange solution was added to this cuvette, and the absorbance was recorded. This served as the control. The Cu-amylin complex was reduced by ascorbic acid under anaerobic conditions (observed by EPR spectroscopy), followed by their reoxidation by

2. EXPERIMENTAL SECTION 2.1. Materials. All reagents were of the highest grade commercially available and were used without further purification. Human amylin peptides, amylin(1−37), amylin(1−19), and all of their mutants were purchased from Zhejiang Ontores Biotechnologies Co., Ltd. (Zhejiang, China), with >95% purity (Scheme 1). Copper sulfate (CuSO4), Xylenol Orange, and the buffers were purchased from Sigma. Hydrogen peroxide (H2O2; 30%), ascorbic acid, sodium hydroxide (NaOH), and sulfuric acid (H2SO4) were purchased from Merck. 2.2. Sample Preparation. An Amylin(1−37) solution was prepared in a 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.0. The truncated amylin peptide stock solutions were prepared in a 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at pH 7.0. A CuSO4 solution was prepared in deionized water. Peptide stock solutions were 0.5 mM, and the CuSO4 stock solution was 5 mM. Cu-amylin complexes were prepared by incubating 1 equiv of amylin with 0.8 equiv of a copper solution for ∼15 min. For the pH perturbation study, the pH of the solution was B


Article

Inorganic Chemistry dioxygen (O2; followed by EPR spectroscopy). A total of 20 μL of a reoxidized solution was added to the cuvette containing the control. The value of the absorbance of the above solution (after subtraction of the control) at 560 nm, when plotted on the calibration curve, yielded the corresponding H2O2 concentration.14

absorption spectrum is identical with that of Cu(II)-bound amylin(1−37) (Figure S4). The bands observed in the region of 250−350 nm are generally assigned as ligand-to-metal charge-transfer bands. The intensity of the broad band at 550 nm is very weak (molar extinction coefficient ε = 120 M−1 cm−1) and can be assigned as ligand-field transitions. Amylin(1−19) peptide in solution displays an intense negative CD band at 235 nm (Figure 2B). Upon the addition of Cu(II), the intensity of this band greatly reduces, producing a CD spectrum with a positive band at 208 nm and a negative band centered at 232 nm, which are not consistent with the formation of α-helix or β-sheet conformation but characteristic of structured loops and turns. A sharp negative band at 279 nm (35831 cm−1) and a positive band at 317 nm (31567 cm−1) are observed (Figure 2B). All of the bands between 200 and 350 nm can be assigned as ligand-to-metal charge-transfer bands. The broad maxima centered at 550 nm in the absorption spectrum get resolved as a weak positive band at 475 nm and a broad negative band at 565 nm, suggesting the presence of ligand-field transitions in the broad absorption maxima at 550 nm (Figure 2A). 3.2. Stoichiometry of the Binding of Cu(II) with Amylin. To determine the number of Cu binding sites in amylin, EPR spectra of the Cu-amylin(1−19) complex with different equivalents of Cu(II) were obtained. As the concentration of Cu(II) is gradually increased to 0.8 equiv, an increase in the intensity of the EPR hyperfine features characteristic of the Cu-amylin complex is observed (following the black arrow in Figure 3A). Above 0.8 equiv, the EPR

3. RESULTS 3.1. Cu(II) Binding to Amylin. EPR Spectroscopy. The EPR spectrum of Cu(II)-bound amylin(1−37) at pH 8.0 (Figure 1, blue) is distinctly different from free Cu(II) in

Figure 1. EPR spectra of CuSO4, Cu-amylin(1−37), and Cuamylin(1−19) with 0.8 equiv of Cu(II) at pH 8.0 in a 10 mM MES buffer.

solution at the same pH. The EPR signal of Cu-amylin(1−37) with g∥ = 2.17 and A∥ = 195 G is consistent with those of tetragonal Cu proteins having a dx2−y2 ground state in a squareplanar or square-pyramidal geometry with a weak axial ligand. The EPR spectra of Cu bound to the truncated amylin(1−19) at pH 8.0 (Figure 1, red) and at other pH values are identical with that of Cu-amylin(1−37) (Figure S1). Moreover, the CV data of Cu-bound amylin(1−19) and amylin(1−37) show identical redox behavior at this pH (Figure S2). Azide is very often used for ligand binding experiments to probe the activesite environment of Cu proteins. Azide binds to Cu-amylin(1− 37) in the presence of excess azide concentration and produces identical absorption and EPR spectra of the azide-bound complex with Cu-amylin(1−19) (Figure S3). This indicates that the Cu binding residues are present in this truncated peptide sequence. Hence, amylin(1−19) has been used through the rest of the manuscript. Absorption and CD Spectroscopy. The binding of Cu to amylin at pH 8.0 has been characterized using absorption and CD spectroscopy. Amylin(1−19) has no well-defined absorption band in the UV−vis region. Cu-amylin(1−19) displays an intense band near 260 nm and a broad band having maxima at 550 nm in the absorption spectrum (Figure 2A). The

Figure 3. (A) EPR spectra of amylin(1−19) in the presence of different equivalents of Cu(II) at pH 8.0 in a 100 mM HEPES buffer. (B) Titration curve for amylin(1−19) with different equivalents of Cu(II) obtained from EPR spectra (plotted at 2900 G, following the black arrow).

hyperfine features characteristic of free Cu(II) in solution starts to grow in (red arrow in Figure 3A). In fact, the difference EPR spectrum of 0.8 equiv of Cu-amylin from Cu-amylin with 2 equiv of Cu(II) shows only free Cu(II) in solution (Figure S5). When the normalized EPR intensity is plotted at 2900 G against equivalents of Cu, saturation is observed at 1 equiv of Cu (Figure 3B). This indicates that Cu(II) binds human amylin in 1:1 stoichiometry to form the Cu-amylin complex. 3.3. pH Perturbations. EPR Spectroscopy. EPR spectra were recorded for Cu-amylin in the pH range of 5−11 (Figure 4A). A pure single species is observed at pH 8.0 with g∥ = 2.17 and A∥ = 195 G (Figure 4A) and has been assigned as component I (or comp I). As the pH is gradually lowered from 8.0 to 6.0, another signal with a distinct set of hyperfine features appears (Figure 4B) with g∥ = 2.20 and A∥ = 162 G, suggesting the presence of another species distinct from component I.

Figure 2. (A) Absorption and (B) CD spectra of Cu-amylin(1−19) at pH 8 in a 20 mM HEPES buffer. C


Article

Inorganic Chemistry

Figure 6. (A) EPR spectra of Cu-amylin(1−19) from pH 6.0 to 5.0. (B) EPR spectra of Cu-amylin(1−19) at pH 5.35, spectrum of component II (comp II from Figure 5B), and the difference spectra of pH 5.35 and a percentage of component II (comp II).

(Figure 7A). The hyperfine features observed at this pH are broad in nature because of the presence of residual component Figure 4. (A) EPR spectra of Cu-amylin(1−19) at different pH values from pH 5.0 to 11.0. The EPR hyperfine features of Cu-amylin(1−19) complexes in the pH ranges of (B) 6.0−8.0 and (C) 5.0−6.0.

This new species has been assigned as component II or comp II (Figure 4B). The intensity of the hyperfine features of component II decreases if the pH is lowered below 6.0 (Figure 4C). When the intensity of the hyperfine features of component II is plotted against pH, it is observed that the relative population of component II is maximum at pH 6.0 (Figures 4C and 5A). The EPR spectrum of the Cu-amylin

Figure 7. (A) EPR spectra of Cu-amylin(1−19) at pH 5.0 and below. (B) EPR spectra of components I−III.

III. The hyperfine features of this new species are distinct from those of free Cu(II) at pH 4.0, demonstrating that Cu is not detached from the peptide scaffold (Figure 7A). This new species is assigned as component IV or comp IV. Upon further lowering of the pH below 4.0, a contribution from free Cu(II) appears (Figure S7). When the pH of the Cu-amylin complex is increased to pH 9.0 and above, a species appears with g∥ = 2.15 and A∥ = 207 G (Figure 4A). It is likely that backbone amides get deprotonated at high pH and coordinate to Cu. In fact, such species have been observed in Cu proteins or peptide complexes at high pH. This high pH (pH 9 and above) species has not been dealt with in this study. Thus, depending on the pH of the solution, Cuamylin shows the presence of four components (I−IV) with distinguishable EPR parameters (Figure 7B and Table 1). All of these components are formed reversibly upon changes in the pH. The EPR parameters of these components having g∥ > ge > 2.0023 are consistent with the presence of D4h Cu systems with a dx2−y2 ground state. From ligand-field theory, the metal hyperfine coupling constant can be expressed as A∥= Pd[−κβ2 − 4/7β2 + (g∥ − ge) + 3/7(g⊥ − ge)], where Pd[Cu2+] = 400 ×

Figure 5. (A) Intensity of the hyperfine of component II (comp II) at different pH values (at 3030 G). (B) EPR spectra of Cu-amylin(1−19) at pH values of 6.5 and 8.0 and the difference spectrum (6.5−8.0) with a percentage of component I (comp I).

complex at pH 6.5 is a mixture of components I and II. Subtraction of a fraction of component I from the EPR spectrum at pH 6.5 generates the EPR spectrum of pure component II (Figure 5B). Upon further lowering of the pH below 6.0, the intensity of the hyperfine features of component II decreases (Figure 4C) and a new species grows in with the EPR parameters of g∥ = 2.26 and A∥= 161 G (Figure 6A), which are distinct from those of both components I and II. This new species has been assigned as component III or comp III. Component III shows a maximum population at pH 5.0 (Figure S6). At pH 5.35, the EPR spectrum is a mixture of components II and III. A pure EPR spectrum of pure component III can be obtained upon subtraction of a fraction of pure component II from the EPR spectrum at pH 5.35 (Figure 6B). Upon further lowering of the pH below 5.0, another new signal starts to appear with A∥ = 145 G and g∥ = 2.31 at pH 4.0

Table 1. EPR Parameters of the Four pH-dependent Components of the Cu-amylin Complex

D

component

pH

g∥

g⊥

A∥ (G)

β2

I II III IV

8.0 6.0 5.0 4.0

2.17 2.20 2.26 2.31

2.03 2.04 2.05 2.05

195 162 161 145

0.635 0.592 0.65 0.69


Article

Inorganic Chemistry 10−4 cm−1, κ[Cu2+] = 0.43, and g⊥ = (gx + gy)/2. The coefficient β2 measures the dx2−y2 character of the ground state. The lower the value of β2, the lower the dx2−y2 character in the ground state, which indicates a higher covalency. The β2 values obtained for components IV, III, II, and I are 0.69, 0.65, 0.592, and 0.635, respectively (Table 1). This indicates that component II is most covalent with 59.2% spin density on the metal and 40.8% spin density on the ligands. Absorption and CD Spectroscopy. The absorption spectra show discrete changes in the charge-transfer and d−d bands upon changes in the pH. At pH 5.0 and 6.0 (predominantly components III and II, respectively), a broad band is observed at 300 nm in the charge-transfer region of the absorption spectra (Figure 8A). As the pH increases, the intensity of this

band at 315 nm.41,42 Thus, the presence of a charge-transfer band at 317 nm likely suggests that deprotonated amide coordination is present in the Cu-amylin complex (Figure 9) for components II and I. Therefore, deprotonated amide is one of the coordinating residues in component II (dominating at pH 6.0) and component I (dominating at pH 8.0) but not in components III and IV. The origin of the negative CD band at 279 nm has been discussed in the Role of N-Terminal Amine section. At pH 5.0 (predominantly component III), a weak negative d−d band at 630 nm and a positive band at 500 nm are observed (Figure 9). At pH 6.0 (predominantly component II), the negative CD band gets blue-shifted to 600 nm, which further shifts to 580 nm at pH 7 (Figure 9B). At pH 8.0, where component I predominates, the negative band gets further blueshifted to 565 nm and the positive band appears at 475 nm. Such high-energy d−d bands of components I and II suggest the presence of a strong ligand field around the Cu center43 and is consistent with the presence of amidate coordination in components I and II. A simultaneous fitting of the absorption and CD spectra shows that the broad d−d bands of component I could be resolved into three transitions at 15895 cm−1 (629 nm), 17938 cm−1 (557 nm), and 20852 cm−1 (479 nm) (Figure 10A,C,

Figure 8. Absorption spectra of Cu-amylin(1−19) (A) in the highenergy region and (B) in the low-energy region at different pH values .

charge-transfer band decreases and is almost absent at pH 8.0 (predominantly component I). This suggests that the ligand involved in the charge-transfer transition at low pH is absent at high pH (predominantly component I). The origin of this charge-transfer band will be ascertained by the CD data on sitedirected mutants. The absorption spectra lack any intense charge-transfer bands in the visible region in the pH range of 4.0−9.0. The weak ligand-field transition bands (Figure 8B) increase in intensity with an increase in the pH. At pH 6.0, a broad maximum is observed near 560 nm, which shifts to 550 nm at pH 8.0 (Figure 8B). When the pH is further increased, the maximum shifts to 520 nm at pH 11.0 (Figure S8). The blue shift of the absorption maximum with an increase in the pH indicates an increased ligand field around the Cu center at high pH. There is a strong negative CD band at 279 nm and a positive CD band at 317 nm (Figure 9), between pH 6.0 and 8.0 (predominantly components II and I). Previous studies on Cubound peptide complexes show that the coordination of deprotonated backbone amide to Cu produces a charge-transfer

Figure 10. (A) Gaussian analysis of the d−d bands of Cu-amylin(1− 19) in the absorption (A and B) and CD (C and D) spectra at pH 8.0 and 6.0 using PeakFit, version 4.12.

bands 1−3). Note that the fourth ligand-field transition could not be resolved and could possibly be at a lower energy. The charge-transfer region in the CD spectra has one positive band at 31567 cm−1 (317 nm) and a negative band at 35831 cm−1 (279 nm) (Figure 11A, bands 4 and 5). The band at 31567 cm−1 (317 nm) can be assigned as a deprotonated amide to Cu charge transfer. At pH 6.0 (component II), there are three d−d transitions at 15698 cm−1 (637 nm), 17662 cm−1 (566 nm), and 20049 cm−1 (499 nm) (Figure 10B,D, bands 1−3). The charge-transfer region shows transitions at 30881 cm−1 (324 nm), 35633 cm−1 (280 nm), and 38892 cm−1 (257 nm) (Figure 11B, bands 4−6). Note that the fourth ligand-field transition could not be observed and could exist at a lower energy (below 12000 cm−1) in component II as well. The CD spectra for components III and IV (observed at pH 5.0 and 4.0, respectively) were not analyzed via a Gaussian distribution

Figure 9. CD spectra of Cu-amylin(1−19) at different pH values (A) in the full range and (B) in the d−d band region. E


Article

Inorganic Chemistry

Figure 11. Gaussian analysis of the CD spectra of Cu-amylin(1−19) (A) at pH 8.0 and (B) at pH 6.0 using PeakFit, version 4.12.

Figure 13. (A) EPR spectra of Cu-NAc-amylin(1−19) at pH 6.0 and 8.0 and the difference spectrum of a percentage of the pH 6.0 EPR spectrum from the pH 8.0 EPR spectrum (pH 8−6), showing the species formed by Cu-NAc-amylin(1−19) at high pH. (B) CD spectra of Cu-amylin(1−19) and Cu-NAc-amylin(1−19) at pH 8.0.

because there are significant contributions of components II and IV in component III at pH 5.0 and component III in component IV at pH 4.0. 3.4. Site-Directed Mutagenesis. Role of N-Terminal Amine. Cu-bound N-terminal acylated amylin, i.e., Cu-NAcamylin(1−19) produces EPR spectra identical with those of Cu-amylin(1−19) at pH 4.0 and 5.0 (Figure S9), suggesting that N-terminus amine is not involved in coordination to the Cu center in components III and IV. At pH 6.0, Cu-NAcamylin(1−19) shows an EPR spectrum (Figure 12A) identical

Cu-NAc-amylin(1−19), implying that the origin of this band is due to N-terminal NH2 to Cu(II) charge transfer (Figures 11, 13B, and S11B) and is present in components II and I. Thus, in addition to an amidate ligand (Absorption and CD Spectroscopy section), components I and II also have an N-terminal amine as a ligand. Role of the Histidine Residue. A histidine residue is present at the 18th position in amylin. To observe the role of His18, a truncated peptide amylin(1−17) has been used. At pH 4.0, Cuamylin(1−17) displays a spectrum identical with that of Cuamylin(1−19), indicating that component IV does not have any histidine coordination (Figure S13). The EPR spectrum at pH 5.0 displays a mixture of species, one being component IV with a g∥ value of 2.31 and a A∥ value of 145 G (assigned with black lines in Figure 14A) and another being a new species with g∥ =

Figure 12. (A) EPR spectra of Cu-NAc-amylin(1−19) at pH 6.0 and component III of Cu-amylin(1−19). (B) EPR spectra of Cu-NAcamylin(1−19) at pH 6.0, 8.0, and 9.5 in 100 mM HEPES and a comparison with components I and II of Cu-amylin(1−19).

with that of component III of Cu-amylin(1−19) [component III of Cu-amylin(1−19) is dominating at pH 5.0]. As the pH is raised above 6.0, the Cu-NAc-amylin(1−19) complex produces a new species at pH 9.5 with g∥ = 2.20 and A∥ = 168 (G) and a pKa of 8.0 (Figures 12B and S10) and does not produce characteristic EPR signal of either component II or I (Figure 12B). The CD spectra of Cu-NAc-amylin(1−19) also support the above observations and do not produce the CD features of components I and II (Figure S11). The EPR spectrum of CuNAc-amylin(1−19) at pH 8.0 is a mixture, and subtraction of the EPR spectrum of pH 6.0 (component III of Cu-NAcamylin(1−19) is formed at pH 6.0) from that of pH 8.0 produces an EPR spectrum of the new species with EPR parameters g∥ = 2.20 and A∥ = 168 G (Figure 13A). The hyperfine features of the new species are completely different from those of components I and II of Cu-amylin(1−19) (Figure S12). This likely implies that the N-terminal NH2 group is one of the coordinating ligands in components I and II. Further, the charge-transfer region of the CD spectrum of Cu-amylin(1−19) shows an intense negative CD band at 280 nm at pH 6.0 and above (Figures 9, 11, and 13B). This intense charge-transfer band at 280 nm is absent in the CD spectrum of

Figure 14. (A) EPR spectra of Cu-amylin(1−17) and Cu-amylin(1− 19) at pH 5.0. (B) EPR spectra of Cu-amylin(1−17) at different pH values.

2.25 and A∥ = 158 G (assigned with red lines in Figure 14A), not observed for native Cu-amylin(1−19). With a further increase in the pH, an EPR signal appears with g∥ = 2.17 and A∥ = 195 G at pH 8.0 (Figures 14B and S14), which resembles component I of Cu-amylin(1−19) (Figure 15A). The CD spectrum also shows the formation of component I in the absence of histidine (Figure 15B). However, interestingly components II and III are not formed in the absence of His18. Further the Cu-bound complex of a histidine mutant of amylin(1−19), i.e., Cu-H18A-amylin(1−19), also shows the formation of component I at pH 8.0 and follows a pH perturbation pattern similar to that of Cu-amylin(1−17) (Figure S15). This clearly indicates that histidine is one of the coordinating ligands in components II and III but not in components I and IV. F


Article

Inorganic Chemistry

and II. The decrease in the redox potential with an increase in the pH suggests the presence of an electron-rich Cu center in component I compared to component II. Thus, Cu-amylin can easily be reduced under physiological conditions by vitamin C, glutathione, and NADH. 3.6. PROS. Reduced Cu-amylin complexes can react with O2 to generate PROS. The amount of PROS formed by Cuamylin(1−19) has been measured via xylenol orange assay (Figures 18A and S18). Amylin(1−19) does not produce any Figure 15. (A) EPR and (B) CD spectra of Cu-amylin(1−17) and Cuamylin(1−19) at pH 8.0.

Role of Disulfide Linkage. Amylin(1−19) contains a disulfide linkage between two cysteine residues at the second and seventh positions. The absence of any intense chargetransfer bands in the low-energy region of the absorption spectra likely rules out the possibility of Cys−S coordination to Cu(II) (Figure 8B). Removal of the disulfide linkage by incorporating Gly at the position of Cys results in the formation of pure component I at pH 9.0 with identical EPR parameters of component I of Cu-amylin(1−19) (Figure 16A).

Figure 18. (A) Amount of PROS produced by Cu-amylin(1−19) at different pH values. (B) Scheme for O2 reduction with the Cu(I) center.

PROS in this assay. At physiological pH, Cu-amylin(1−19) produces ∼40% of PROS, indicating the reduction of O2 via a one-electron pathway and thus producing less than 50% PROS (Figure 18B). At pH 5.0, 6.0, and 8.0, where components III, II, and I dominate, Cu-amylin(1−19) produces almost a similar amount of PROS (Figure 18A).

4. DISCUSSION Active-Site Environment. The Cu-amylin complexes and site-specific mutants have been probed with absorption, CD, and EPR spectroscopy. The results indicate that Cu binds human amylin in a 1:1 ratio and the binding domain lies within the first 19 amino acid residues. A pH perturbation study shows that there are four species between pH 4.0 and 8.0 (Figure 7B and Table 1). A band at 38892 cm−1 (257 nm, band 6 in Table 2) is observed in the charge-transfer region of the absorption

Figure 16. (A) EPR spectra of Cu-2,7-Gly-amylin(1−19) at pH 9.0 and Cu-amylin(1−19) at pH 8.0. (B) EPR spectra of component II of Cu-amylin(1−19) and Cu-2,7-Gly-amylin(1−19) observed at pH 7.0.

The highest population of component II is observed at pH 7.0 (Figures 16B and S16). The pKa values for the formation of components II and I are 6.5 and 8.1, respectively, relative to those of 5.5 and 6.6, respectively, for the native amylin peptide (Figure S16 and Figure 5A). This suggests that the removal of the disulfide linkage only results in an increase of the pKa value of the pH-dependent species and is possibly not involved in coordinating directly to Cu. 3.5. CV. The CV data of Cu-amylin(1−19) at pH 5.0−9.0 show a quasi-reversible process with E1/2 = 241 mV vs NHE at pH 7. The E1/2 value decreases with an increase in the pH (Figures 17 and S17). The largest change in E1/2, ∼43 mV, is observed between pH 6.0 and 8.0, i.e., between components I

Table 2. Charge-Transfer and d−d Bands of Cu-amylin at pH 6.0 and 8.0 no.

band

1 2 3 4 5 6

d→d d→d d→d Namidate− → Cu NH2 → Cu N-His → Cu

pH 8.0 15895 17938 20852 31567 35831

(629 (557 (479 (317 (279

pH 6.0 nm) nm) nm) nm) nm)

15698 17662 20049 30881 35633 38892

(637 (566 (499 (324 (280 (257

nm) nm) nm) nm) nm) nm)

and CD spectra (Figure 8A and band 6 in Figure 11B) in component II. Coordination of the imidazole nitrogen atom of histidine gives rise to two π-to-Cu(II) charge-transfer transitions near 260 and 330 nm.44,45 Hence, the band at 38892 cm−1 (257 nm, Table 2) might be derived from a NHis(π) (His18)-to-Cu charge transfer. Moreover, this band at 38892 cm−1 (257 nm) is absent in the CD spectrum of component I, which does not have any histidine coordination (Figure 11A). The presence of a deprotonated amide-to Cu charge-transfer band (at 30881 cm−1, 324 nm; Table 2) in the

Figure 17. E1/2 potential (vs SHE) of the Cu-amylin(1−19) complex at different pH values. G


Article

Inorganic Chemistry Table 3. Charge-Transfer Bands, EPR Parameters, and E1/2 Values of the Four Components of Cu-amylin(1−19) charge transfer component

amidate (N−) → Cu

amine (NH2) → Cu

components formed by histidine mutant

g∥

A∥ (G)

β2

E1/2 (mV vs SHE)

I II III IV

yes (317 nm) yes (324 nm) no no

yes (279 nm) yes (280 nm) no no

yes no no yes

2.17 2.20 2.26 2.31

195 162 161 145

0.63 0.59 0.65 0.69

+206 +249 +246 +257

negative charge from amidate, making total 1+ charge over the Cu center) as component II. β2 increases gradually from component II to component IV, consistent with a slight increase in E1/2 indicating a gradual weakening of the ligand field around Cu. Component III is produced by Cu-NAcamylin(1−19), but the EPR is perturbed upon histidine mutation, which suggests that histidine is the only N-donor ligand in component III. Other ligands are likely to arise from carbonyl or water molecules, forming a square-planar or squarepyramidal geometry. The only discernible change between components III and IV is the loss of the histidine ligand in component IV. The current set of data does not indicate coordination by side groups of other amino acids for component IV, and these ligands are likely to be amide carbonyls or water. It is tempting to use empirical correlations between the g and A values to assign coordination environments for peptide-bound Cu complexes.37,50,51 Such an exercise would result in descriptions (Table 4) that are consistent with

CD spectrum (Figure 11B) confirms the presence of deprotonated amide coordination in component II. Thus, component II has a charge-transfer band from an amidate at 324 nm and a charge-transfer band from the N-terminal amine at 280 nm, and its EPR is perturbed upon histidine mutation. Thus, component II has amidate, N-terminal amine, and histidine coordination as nitrogen-donor ligands. The EPR g∥ and A∥ values yield a β2 value of 0.59, and the high-energy ligand-field transitions (Tables 1 and 3) indicate that the site is likely to have five-coordinate square-pyramidal geometry. Thus, the two other ligations are likely to be occupied by amide carbonyl and or water (O). Backbone amides generally coordinate to the Cu center via carbonyl oxygen at low pH, which upon deprotonation at high pH leads to coordination via a deprotonated amide (N−) center.46,47 In fact, the Cu-amylin complex shows N− coordination at high pH in component I, which supports the carbonyl oxygen coordination at low-pH components (components II, III, and IV; see below). Component I does not have histidine coordination, as indicated by the EPR and CD data (Figure 15A,B). The fact that component I results from deprotonation of component II implies that either a bound water is deprotonated to hydroxide or a bound carbonyl is replaced by an amidate. However, the g∥ value of component I is lower than that of component II, while the A∥ value of the latter is higher. A higher A∥ with a lower g∥ in component I relative to component II is consistent with the loss of a ligand in component I relative to component II. This ligand is, of course, the histidine that is coordinated to the Cu center in component II. Additionally, the CuII/I E1/2 is ∼43 mV more negative in component I at pH 8.0 relative to component II at pH 6.0 (Figures 17 and S17 and Table 3). This implies substantially increased stabilization of the Cu2+ state in component I relative to component II. Taken together, these data suggest that component I likely involves deprotonation of a ligand, leading to stabilization of the Cu2+ state and dissociation of a weak ligand and resulting in increased β2. Thus, the likely coordinating ligands of component I are amidate (N), an N-terminal amine (N), a carbonyl (O), and finally an amidate (N) or hydroxide (HO−) in a square-planar geometry that results from the deprotonation of component II.48,49 Hence, the pKa associated with components I and II might arise from an amide (CO) ↔ −N (amidate) or H2O ↔ −OH equilibrium. In order to form component I from component II, a histidine is displaced and a Cu-amidate(N−) or a Cu−OH bond is formed via deprotonation of carbonyl or water. Considering the chelate effect, a Cu-amidate(N−) bond would be favored over a Cu−OH bond. There should not be much additional entropic cost in replacing the histidine with an amidate from the same peptide chain. Components III and IV result in changes in the ground-state wave function but have very similar Cu2+/+ potentials. Thus, the change of ligation in these components is likely not involved with a change in the charge of the active site, i.e., the overall charge remains 1+ (a 2+ charge over the Cu center and a

Table 4. EPR Parameters of Different pH-Dependent Components of the Cu-amylin Complex and Their Coordination Mode from the Peisach−Blumberg Plot

component

pH

g∥

g⊥

A∥ (G)

β2

coordination mode from the Peisach− Blumberg plot

I II III IV

8.0 6.0 5.0 4.0

2.17 2.20 2.26 231

2.03 2.04 2.05 2.05

195 162 161 145

0.635 0.592 0.65 0.69

4N 3N1O 1N3O 4O

the descriptors obtained from the spectroscopic data of Cuamylin complexes (and mutants). However, it must be noted that these correlations assume a strictly square-planar geometry at the Cu center and are very vulnerable to the total charge at the center. Recently, using a short peptide, amylin(15−22), a 3N1O coordination mode contributed by histidine, two deprotonated amides, and O from serine carbonyl or hydroxide, has been proposed as a key species at pH 7.5.37 Another study using amylin(1−19) demonstrates a 4N coordination mode contributed by histidine and three amidates in a square-planar geometry at pH 6.0 and above.38 However, both models differ from the present observations and cannot be compared because the presence of four distinct pH-dependent components has not been considered in the previous models and they might contain a mixture of species. Moreover, the two models reject terminal amine as the coordinating ligand but consider histidine as the key ligand at physiological pH. However, the present work precisely shows the presence of N-terminal amine in components II and I and a histidine residue in components II and III (but not at pH 8.0 in component I). Components I and II are physiologically relevant species with reduction potentials of 206 and 249 mV vs NHE, respectively H


Article

Inorganic Chemistry (Scheme 2). Thus, these Cu-amylin species can be easily reduced by physiological reductants like vitamin C and

Notes

Scheme 2. Schematic Representation of Coordination Environments of Components I and II

ACKNOWLEDGMENTS We thank CSIR, India [Grant 01(2764)/13/EMR-II], DST, India (Grant EMR/2014/000392), Government of India, and IACS for funding this research. M.S. is thankful to the IACSintegrated Ph.D. program for a Senior Research Fellowship.

The authors declare no competing financial interest.

■ ■

(1) Reaven, G. M. Role of Insulin Resistance in Human Disease. Diabetes 1988, 37, 1595−1607. (2) Diabetes Fact Sheet; World Health Organization, November 2016. (3) Höppener, J. W. M.; Ahrén, B.; Lips, C. J. M. Islet Amyloid and Type 2 Diabetes Mellitus. N. Engl. J. Med. 2000, 343, 411−419. (4) Jaikaran, E. T. A. S.; Clark, A. Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology. Biochim. Biophys. Acta, Mol. Basis Dis. 2001, 1537, 179−203. (5) Cooper, G. J.; Willis, A. C.; Clark, A.; Turner, R. C.; Sim, R. B.; Reid, K. B. Purification and characterization of a peptide from amyloidrich pancreases of type 2 diabetic patients. Proc. Natl. Acad. Sci. U. S. A. 1987, 84, 8628−8632. (6) Westermark, P.; Andersson, A.; Westermark, G. T. Islet Amyloid Polypeptide, Islet Amyloid, and Diabetes Mellitus. Physiol. Rev. 2011, 91, 795−826. (7) Moore, C. X.; Cooper, G. J. S. Co-secretion of amylin and insulin from cultured islet β-cells: Modulation by nutrient secretagogues, islet hormones and hypoglycemic agents. Biochem. Biophys. Res. Commun. 1991, 179, 1−9. (8) Cooper, G. J. S.; Day, A. J.; Willis, A. C.; Roberts, A. N.; Reid, K. B. M.; Leighton, B. Amylin and the amylin gene: structure, function and relationship to islet amyloid and to diabetes mellitus. Biochim. Biophys. Acta, Mol. Cell Res. 1989, 1014, 247−258. (9) Haataja, L.; Gurlo, T.; Huang, C. J.; Butler, P. C. Islet Amyloid in Type 2 Diabetes, and the Toxic Oligomer Hypothesis. Endocr. Rev. 2008, 29, 303−316. (10) Janson, J.; Ashley, R. H.; Harrison, D.; McIntyre, S.; Butler, P. C. The mechanism of islet amyloid polypeptide toxicity is membrane disruption by intermediate-sized toxic amyloid particles. Diabetes 1999, 48, 491−498. (11) Cao, P.; Marek, P.; Noor, H.; Patsalo, V.; Tu, L. H.; Wang, H.; Abedini, A.; Raleigh, D. P. Islet amyloid: from fundamental biophysics to mechanisms of cytotoxicity. FEBS Lett. 2013, 587, 1106−1118. (12) Viles, J. H. Metal ions and amyloid fiber formation in neurodegenerative diseases. Copper, zinc and iron in Alzheimer’s, Parkinson’s and prion diseases. Coord. Chem. Rev. 2012, 256, 2271− 2284. (13) Faller, P.; Hureau, C.; La Penna, G. Metal Ions and Intrinsically Disordered Proteins and Peptides: From Cu/Zn Amyloid-β to General Principles. Acc. Chem. Res. 2014, 47, 2252−2259. (14) Pramanik, D.; Ghosh, C.; Dey, S. G. Heme−Cu Bound Aβ Peptides: Spectroscopic Characterization, Reactivity, and Relevance to Alzheimer’s Disease. J. Am. Chem. Soc. 2011, 133, 15545−15552. (15) Kazi, T. G.; Afridi, H. I.; Kazi, N.; Jamali, M. K.; Arain, M. B.; Jalbani, N.; Kandhro, G. A. Copper, Chromium, Manganese, Iron, Nickel, and Zinc Levels in Biological Samples of Diabetes Mellitus Patients. Biol. Trace Elem. Res. 2008, 122, 1−18. (16) Taylor, C. G. Zinc, the pancreas, and diabetes: insights from rodent studies and future directions. BioMetals 2005, 18, 305−312. (17) Rajpathak, S. N.; Crandall, J. P.; Wylie-Rosett, J.; Kabat, G. C.; Rohan, T. E.; Hu, F. B. The role of iron in type 2 diabetes in humans. Biochim. Biophys. Acta, Gen. Subj. 2009, 1790, 671−681. (18) Tanaka, A.; Kaneto, H.; Miyatsuka, T.; Yamamoto, K.; Yoshiuchi, K.; Yamasaki, Y.; Shimomura, I.; Matsuoka, T.-a.; Matsuhisa, M. Role of Copper Ion in the Pathogenesis of Type 2 Diabetes. Endocr. J. 2009, 56, 699−706.

glutathione. The xylenol orange assay (Figure 18) shows that, at physiological pH, reduced Cu-amylin produces 40% H2O2 during oxidation of the reduced Cu-amylin by O2 (Figure 18B and the Supporting Information). This highlights the possible role of Cu as a stimulator of oxidative stress in T2Dm. The CD data show that coordination of Cu to amylin results in the formation of loops rather than β sheets (Figure 2), which facilitates aggregation. Thus, Cu hinders the formation of β sheets and possibly enhances the formation of an oligomeric species that is potentially more toxic to the β cells of the pancreas than the fibrillar form.25,28 Note that the His18 residue, involved in the coordination of Cu, is absent in rat amylin. Rat amylin is nonamylodogenic and has an Arg18 residue instead of the histidine residue, and rats do not suffer from T2Dm (Scheme 1). This could likely implicate a possible role of Cu-amylin in the development of T2Dm.

5. CONCLUSION In summary, our study has revealed that Cu binds to native human amylin in a 1:1 ratio. The Cu binding domain lies within the truncated amylin(1−19) fragment. The residues involved in the two physiological relevant forms (components I and II) are identified using a combination of spectroscopic tools on sitedirected mutants. The N-terminal amine and His18 are established as ligands to the Cu center along with the backbone amidate. Components I and II at physiological pH (6.0−8.0) can generate H2O2 and potentially induce toxicity in the pancreatic β cells. Cu-amylin hinders the formation of β sheets and possibly enhances the formation of an oligomeric species that is potentially more toxic to the β cells of the pancreas.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b02266. EPR, CD, CV, absorption and azide binding data of Cuamylin(1−19), Cu-amylin(1−37), and relevant mutants; formation and decay of component III; pKa plots; PROS data (PDF)

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Somdatta Ghosh Dey: 0000-0002-6142-2202 I


Article

Inorganic Chemistry (19) Bao, W.; Rong, Y.; Rong, S.; Liu, L. Dietary iron intake, body iron stores, and the risk of type 2 diabetes: a systematic review and meta-analysis. BMC Med. 2012, 10, 1−13. (20) Wilson, J. G.; Lindquist, J. H.; Grambow, S. C.; Crook, E. D.; Maher, J. F. Potential Role of Increased Iron Stores in Diabetes. Am. J. Med. Sci. 2003, 325, 332−339. (21) Zhao, Z.; Li, S.; Liu, G.; Yan, F.; Ma, X.; Huang, Z.; Tian, H. Body Iron Stores and Heme-Iron Intake in Relation to Risk of Type 2 Diabetes: A Systematic Review and Meta-Analysis. PLoS One 2012, 7, e41641. (22) Mukherjee, S.; Dey, S. G. Heme Bound Amylin: Spectroscopic Characterization, Reactivity, and Relevance to Type 2 Diabetes. Inorg. Chem. 2013, 52, 5226−5235. (23) Seal, M.; Mukherjee, S.; Dey, S. G. Fe-oxy adducts of heme-Aβ and heme-hIAPP complexes: intermediates in ROS generation. Metallomics 2016, 8, 1266−1272. (24) Bozkurt, F.; Tekin, R.; Gulsun, S.; Satıcı, Ö .; Deveci, O.; Hosoglu, S. The levels of copper, zinc and magnesium in type II diabetic patients complicated with foot infections. Int. J. Diabetes Dev. Countries 2013, 33, 165−169. (25) Yu, Y.-P.; Lei, P.; Hu, J.; Wu, W.-H.; Zhao, Y.-F.; Li, Y.-M. Copper-induced cytotoxicity: reactive oxygen species or islet amyloid polypeptide oligomer formation. Chem. Commun. 2010, 46, 6909− 6911. (26) Kallay, C.; David, A.; Timari, S.; Nagy, E. M.; Sanna, D.; Garribba, E.; Micera, G.; De Bona, P.; Pappalardo, G.; Rizzarelli, E.; Sovago, I. Copper(II) complexes of rat amylin fragments. Dalton. Trans. 2011, 40, 9711−9721. (27) Ward, B.; Walker, K.; Exley, C. Copper(II) inhibits the formation of amylin amyloid in vitro. J. Inorg. Biochem. 2008, 102, 371−375. (28) Lee, S. J. C.; Choi, T. S.; Lee, J. W.; Lee, H. J.; Mun, D.-G.; Akashi, S.; Lee, S.-W.; Lim, M. H.; Kim, H. I. Structure and assembly mechanisms of toxic human islet amyloid polypeptide oligomers associated with copper. Chem. Sci. 2016, 7, 5398−5406. (29) Sinopoli, A.; Magri, A.; Milardi, D.; Pappalardo, M.; Pucci, P.; Flagiello, A.; Titman, J. J.; Nicoletti, V. G.; Caruso, G.; Pappalardo, G.; Grasso, G. The role of copper(ii) in the aggregation of human amylin. Metallomics 2014, 6, 1841−1852. (30) Kaneto, H.; Katakami, N.; Matsuhisa, M.; Matsuoka, T.-a. Role of Reactive Oxygen Species in the Progression of Type 2 Diabetes and Atherosclerosis. Mediators Inflammation 2010, 2010, 1−11. (31) Masad, A.; Hayes, L.; Tabner, B. J.; Turnbull, S.; Cooper, L. J.; Fullwood, N. J.; German, M. J.; Kametani, F.; El-Agnaf, O. M. A.; Allsop, D. Copper-mediated formation of hydrogen peroxide from the amylin peptide: A novel mechanism for degeneration of islet cells in type-2 diabetes mellitus? FEBS Lett. 2007, 581, 3489−3493. (32) Zraika, S.; Hull, R. L.; Udayasankar, J.; Aston-Mourney, K.; Subramanian, S. L.; Kisilevsky, R.; Szarek, W. A.; Kahn, S. E. Oxidative stress is induced by islet amyloid formation and time-dependently mediates amyloid-induced beta cell apoptosis. Diabetologia 2009, 52, 626−635. (33) Lenzen, S.; Drinkgern, J.; Tiedge, M. Low antioxidant enzyme gene expression in pancreatic islets compared with various other mouse tissues. Free Radical Biol. Med. 1996, 20, 463−466. (34) Tiedge, M.; Lortz, S.; Drinkgern, J.; Lenzen, S. Relation Between Antioxidant Enzyme Gene Expression and Antioxidative Defense Status of Insulin-Producing Cells. Diabetes 1997, 46, 1733− 1742. (35) Evans, J. L.; Goldfine, I. D.; Maddux, B. A.; Grodsky, G. M. Are Oxidative Stress−Activated Signaling Pathways Mediators of Insulin Resistance and β-Cell Dysfunction? Diabetes 2003, 52, 1−8. (36) Cooksey, R. C.; Jouihan, H. A.; Ajioka, R. S.; Hazel, M. W.; Jones, D. L.; Kushner, J. P.; McClain, D. A. Oxidative Stress, β-Cell Apoptosis, and Decreased Insulin Secretory Capacity in Mouse Models of Hemochromatosis. Endocrinology 2004, 145, 5305−5312. (37) Sánchez-López, C.; Cortés-Mejía, R.; Miotto, M. C.; Binolfi, A.; Fernández, C. O.; del Campo, J. M.; Quintanar, L. Copper

Coordination Features of Human Islet Amyloid Polypeptide: The Type 2 Diabetes Peptide. Inorg. Chem. 2016, 55, 10727−10740. (38) Rowinska-Zyrek, M. Coordination of Zn2+ and Cu2+ to the membrane disrupting fragment of amylin. Dalton Trans. 2016, 45, 8099−8106. (39) Lee, E. C.; Ha, E.; Singh, S.; Legesse, L.; Ahmad, S.; Karnaukhova, E.; Donaldson, R. P.; Jeremic, A. M. Copper(II)− human amylin complex protects pancreatic cells from amylin toxicity(). Phys. Chem. Chem. Phys. 2013, 15, 12558−12571. (40) Rivillas-Acevedo, L.; Sánchez-López, C.; Amero, C.; Quintanar, L. Structural Basis for the Inhibition of Truncated Islet Amyloid Polypeptide Aggregation by Cu(II): Insights into the Bioinorganic Chemistry of Type II Diabetes. Inorg. Chem. 2015, 54, 3788−3796. (41) Daniele, P. G.; Prenesti, E.; Ostacoli, G. Ultraviolet-circular dichroism spectra for structural analysis of copper(II) complexes with aliphatic and aromatic ligands in aqueous solution. J. Chem. Soc., Dalton Trans. 1996, 3269−3275. (42) Rasia, R. M.; Bertoncini, C. W.; Marsh, D.; Hoyer, W.; Cherny, D.; Zweckstetter, M.; Griesinger, C.; Jovin, T. M.; Fernández, C. O. Structural characterization of copper(II) binding to α-synuclein: Insights into the bioinorganic chemistry of Parkinson’s disease. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 4294−4299. (43) Rosenberg, R. C.; Root, C. A.; Bernstein, P. K.; Gray, H. B. Spectral studies of copper(II) carboxypeptidase A and related model complexes. J. Am. Chem. Soc. 1975, 97, 2092−2096. (44) Fawcett, T. G.; Bernarducci, E. E.; Krogh-Jespersen, K.; Schugar, H. J. Charge-transfer absorptions of copper(II)-imidazole and copper(II)-imidazolate chromophores. J. Am. Chem. Soc. 1980, 102, 2598−2604. (45) Pantoliano, M. W.; Valentine, J. S.; Nafie, L. A. Spectroscopic studies of copper(II) bound at the native copper site or substituted at the native zinc site of bovine erythrocuprein (superoxide dismutase). J. Am. Chem. Soc. 1982, 104, 6310−6317. (46) Holm, R. H.; Kennepohl, P.; Solomon, E. I. Structural and Functional Aspects of Metal Sites in Biology. Chem. Rev. 1996, 96, 2239−2314. (47) Sigel, H.; Martin, R. B. Coordinating properties of the amide bond. Stability and structure of metal ion complexes of peptides and related ligands. Chem. Rev. 1982, 82, 385−426. (48) Jacobson, F.; Pistorius, A.; Farkas, D.; De Grip, W.; Hansson, Ö .; Sjölin, L.; Neutze, R. pH dependence of copper geometry, reduction potential, and nitrite affinity in nitrite reductase. J. Biol. Chem. 2007, 282, 6347−6355. (49) Kobayashi, K.; Tagawa, S.; Suzuki, S.; Deligeer. The pHdependent changes of intramolecular electron transfer on coppercontaining nitrite reductase. J. Biochem. 1999, 126, 408−412. (50) Drew, S. C.; Ling Leong, S.; Pham, C. L. L.; Tew, D. J.; Masters, C. L.; Miles, L. A.; Cappai, R.; Barnham, K. J. Cu2+ Binding Modes of Recombinant α-Synuclein − Insights from EPR Spectroscopy. J. Am. Chem. Soc. 2008, 130, 7766−7773. (51) Peisach, J.; Blumberg, W. Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins. Arch. Biochem. Biophys. 1974, 165, 691−708.

J


Active-Site Environment of Copper-Bound Human ... - ACS Publications

Recommend Documents