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. 2011 Jul 8;410(2):294-306.
doi: 10.1016/j.jmb.2011.05.015. Epub 2011 May 17.

A two-site mechanism for the inhibition of IAPP amyloidogenesis by zinc

Samer Salamekh  1 , Jeffrey R Brender, Suk-Joon Hyung, Ravi Prakash Reddy Nanga, Subramanian Vivekanandan, Brandon T Ruotolo, Ayyalusamy Ramamoorthy
Affiliations

A two-site mechanism for the inhibition of IAPP amyloidogenesis by zinc

Samer Salamekh et al. J Mol Biol. .

Abstract

Human islet amyloid polypeptide (hIAPP) is a highly amyloidogenic protein co-secreted with insulin in response to glucose levels. The formation of hIAPP amyloid plaques near islet cells has been linked to the death of insulin-secreting β-cells in humans and the progression of type II diabetes. Since both healthy individuals and those with type II diabetes produce and secrete hIAPP, it is reasonable to look for factors involved in storing hIAPP and preventing amyloidosis. We have previously shown that zinc inhibits the formation of insoluble amyloid plaques of hIAPP; however, there remains significant ambiguity in the underlying mechanisms. In this study, we show that zinc binds unaggregated hIAPP at micromolar concentrations similar to those found in the extracellular environment. By contrast, the fibrillar amyloid form of hIAPP has low affinity for zinc. The binding stoichiometry obtained from isothermal titration calorimetry experiments indicates that zinc favors the formation of hIAPP hexamers. High-resolution NMR structures of hIAPP bound to zinc reveal changes in the electron environment along residues that would be located along one face of the amphipathic hIAPP α-helix proposed as an intermediate for amyloid formation. Results from electrospray ionization mass spectroscopy investigations showed that a single zinc atom is predominantly bound to hIAPP and revealed that zinc inhibits the formation of the dimer. At higher concentrations of zinc, a second zinc atom binds to hIAPP, suggesting the presence of a low-affinity secondary binding site. Combined, these results suggest that zinc promotes the formation of oligomers while creating an energetic barrier for the formation of amyloid fibers.

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Figures

Figure 1
Figure 1
Amino acid sequences of the peptides used. All unlabelled peptides are amidated and contain oxidized cysteines. The imidazole of His 18 can bind to zinc and the substitution of arginine for histidine in rat IAPP removes a potential zinc binding site.
Figure 2
Figure 2
Heat evolved from the titration of ZnCl2 into hIAPP1–19 with 100 mM Tris buffer and (A) 100 μM NaCl, (B) 100 μM NaCl with 30%TFE, and with (C) 100 mM NaCl. The data was fit to a single binding site model and the apparent thermodynamic parameters can be found in Table 1. All ITC experiments were conducted at pH 7.3 and 25°C
Figure 3
Figure 3
Heat evolved from the titration of ZnCl2 into buffer (100 mM Tris buffer and 100μM NaCl), hIAPP1–19, and MSI-361 under identical conditions. The heat evolved from the interaction between MSI-361 and zinc is comparable in magnitude to the control, and significantly less than the interaction between hIAPP1–19 and zinc. Since both peptides are 19 residues long and contain one histidine, the results indicate that the observed interaction between hIAPP and zinc are sequence specific.
Figure 4
Figure 4
Heat evolved from the titration of ZnCl2 into hIAPP1–19 at pH 7.3 and 25°C, as measured by ITC. In solution with 100 mM Tris buffer and 100 μM NaCl ( formula image), zinc has one apparent binding site at sub- stoichiometric ratios of 0.136 zinc to 1 hIAPP1–19. In the presence of 30% TFE ( formula image) the peptide adopts an α-helical conformation, causing a shift in the apparent binding stoichiometry of the primary binding site to 0.24 zinc to 1 hIAPP1–19 and the addition of an endothermic process at higher concentrations of zinc.
Figure 5
Figure 5
Addition of hIAPP1–37 to 2.5 μM of the fluorescence zinc sensor, FluoZin-1, and 2.5 μM zinc. A decrease in fluorescence intensity corresponds to zinc being displaced from the zinc sensor and binding to the peptide. The solution was initially maintained at 4 °C to prevent aggregation. At the 6 μM titration point the temperature was raised to 25 °C. The peptide was allowed to stand at the 8 μM titration point until aggregation became evident by the turbidity of the solution. The increase in fluorescence intensity at 8 μM indicates the zinc is being displaced from the peptide by fibrillization. The decrease in fluorescence after addition of fresh peptide confirms the sensor is still binding zinc after fibrillization.
Figure 6
Figure 6
ESI-MS spectra of oxidized (A, C) and reduced (B, D) hIAPP1–37 in absence (A, B) of zinc and presence (C, D) of 1 mM zinc. In the absence of zinc, a +2 oxidized monomer peak is observed at 1952 m/z (A) and a +2 reduced monomer peak is observed at 1954 m/z (B). A minor +3 dimer peak is also observed at 2604 m/z for oxidized IAPP and 2605 m/z for reduced IAPP. The addition of zinc to oxidized IAPP (C) reveals +2 monomer peaks at 1984 m/z and 2017 m/z, corresponding to monomers bound to 1 and 2 zinc ions respectively. Similar +2 monomer peaks were observed at 1985 m/z and 2018 m/z with the addition of zinc to the reduced peptide (D). Compared to oxidized hIAPP, a greater proportion of reduced hIAPP is bound to 1 and 2 zinc ions, indicating the presence of non-native interactions with cysteine after reduction of the disulfide bond. No peaks were observed near 2604 m/z with the addition of zinc.
Figure 7
Figure 7
(A) Concentration dependence of zinc binding of 10 μM hIAPP1–37 by ESI-MS. The first binding site is saturated at 10 μM ZnCl2, while a second binding site becomes evident at 1000 μM ZnCl2. (B) Comparison of experimental values to a fit of the data according to a two site independent binding model. A close-up of the low zinc concentration values is shown in the inset. Kd values from the fit were calculated to be 2 ± 0.3 μM and 1200 ± 300 μM.
Figure 8
Figure 8
Overlay of SOFAST-HMQC spectra of 15N-hIAPP1–37 in the presence (blue) and absence (red) of zinc chloride at pH 5. The greatest chemical shift occurs at His 18; however, Thr 6, Arg 11, and Leu 12 display significant chemical shifts as well.
Figure 9
Figure 9
Overlay of the 2D 1H-1H TOCSY spectra of hIAPP1–19 in solution and a select region of the NOESY spectra in the absence (red) and presence (blue) of 10 mM ZnCl2. Addition of zinc causes downfield shifts for the HN proton residues of Cys 7, Glu 10, Ala 13, Leu 16, Val 17 and upfield shifts for residues Arg 11 and His 18. A significant up-field shift of 0.2 ppm for the Hδ2 proton of His 18 is observed upon the addition of zinc (shown in the NOESY strip).
Figure 10
Figure 10
Changes in Hα and HN chemical shifts of hIAPP1–19 upon binding to zinc. Also shown are side chain chemical shift differences of Thr 6 (Hβ) and His 18 (Hβ2/3 and Hδ2). Noticeable differences are localized at Thr 6, Cys 7, Leu 16, Val 17 and His 18.
Figure 11
Figure 11
High-resolution NMR structures of hIAPP1–19 in the absence (left) and in the presence (right) of 10 mM ZnCl2. Zinc binding induces ordering of the secondary structure at the N-terminus. In the absence of zinc, Gln 10, Asn 14, His 18 are oriented in same plane whereas Asn 14 and His 18 are in the same plane with the His 18 ring flipped horizontally while Gln 10 is moved closer to the N-terminus.
Figure 12
Figure 12
Diagram of the fibrillization pathway in the presence of zinc, not drawn to scale. In the presence of zinc, the formation of off-pathway hexameric species is stabilized while the formation of dimers is inhibited. Since zinc does not appreciably interact with the mature fibril, the dissociation of zinc poses a large thermodynamic barrier for the formation of mature fiber.
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