Zn(II) as a minimal chaperone mimic to retard Aβ peptide fibril formation Astrid Gräslund Department of Biochemistry and Biophysics Stockholm University Lorentz workshop, Leiden, April 17, 215 Potential aggregation process of Aβ peptide: disordered aggregate off-pathway β-hairpin β-structured oligomer mature fibril monomer disordered aggregate on-pathway Review: Wärmländer et al., ChemBioChem (213) 14, 1692-174 pre-fibrillar β-structured aggregate
Aβ peptide sequence: DAEFR5HDSGY1EVHHQ15KLVFF2AEDVG25SNKGA3IIGLM35VGGVV4IA42 Interaction with metal ions metal ions, competition between Cu(II) and Zn(II): 1 Fluorescence of Tyr1 in A (1 28) quenched by paramagnetic Cu 2+, in the presence of Zn 2+ (, 6, 9 M) Fluorescence intensity / I/I.8.6 Binding constants: Cu: K D =.36 M at ph 7.2 Zn: K D = 1.1 M at ph 7.2 assuming that both share a common binding site Residuals.4.2.1 1 2 3 -.1 -.2 1 2 Copper Concentration / [µm] 3 Zn 2+ and Cu 2+ interaction with A (1 4) studied by NMR. 1 H 15 N HSQC spectra. Cu paramagnetic broadning/intermediate chemical exchange Zn broadening due to intermediate chemical exchange Similar binding sites in the N terminus for Cu and Zn. Second binding site for Zn? Increased rigidity and slower amide proton exchange after Zn binding. Amide Satbility Change R2 Difference Apparent Affinity / M Relative Signal Intensity.4.2 1. 2. 3. 4. 5. 6. 7. 8. 9. 1. 11. 12. 13. 14. 15. 16. 17. 18. 19. 2. 21. 22. 23. 24. 25. 26. 27. 28. 29. 3. 31. 32. 33. 34. 35. 36. 37. 38. 39. 4. 18 residue 16 14 12 Zn B 1 8 6 4 2 2-2 -4-6 -8-1.4.2 Cu A 1 6 11 16 21 26 31 36 Residue Zn 1 11 21 31 Zn 1 11 21 31 Residue number C D
1 H 13 C HSQC of 5 M A (1 4) before and after addition of Zn. A Tyr1 His6,13,14 but not Tyr1 crosspeaks are affected by Zn. B Tyr1 A His1 3 B His 6 His1 3 His 6 His1 4 His1 4 Model of Zn binding site at the N terminus of A (1 4). K D ~1 M at ph 7.2, similar for Zn and Cu. His6 CH 2 H N N N H N terminus Danielsson et al. (27) FEBS J. 274, 46 59. CH 2 His13 CH 2 His14 Similar to model for Cu binding to A (1 28) by Syme et al., 24, JBC 279: 18169. C terminus
Addition of Cu or Zn to A (1 4) (1:1) decreases the ability to bind ThioflavinT. Thioflavin T fluorescence is enhanced when it binds to amyloid structures. ThT fluorescence 1.2 1.8.6.4.2 lag phase aggregation -.2 5 1 15 2 25 3 35 + Zn + Cu minutes Zn, Cu 1:1 Increased lag phase Disordered aggregates A monomer Concentration, temperature, time etc aggregates (binding ThT)
Cu 2+ and Zn 2+ bind competitively and weakly to A at ph 7.2. Both ions have a relatively well defined major binding site in the N-terminus, involving 3 Histidines and the N-terminus. Effect of metal binding at lower ph? Ghalebani et al., Biochem. Biophys. Res. Comm. 421 (212) 554-6. Effects of Zn(II) ion binding at low stochiometric ratios molecular details Kinetic studies by ThT fluorescence Structural and thermodynamic studies by high resolution NMR Abelein A., Gräslund, A. Danielsson, J. Zinc as chaperone mimicking agent for retardation of amyloid β peptide fibril formation. Proc. Nat. Sci. US (in press) 215. ADAM 212
Modulation of A aggregation by Zn 2+ ions ThT aggregation kinetics at different A concentrations from 1 (red) to 2 (violet) µm Zn 2+ at low conc. retards A fibril formation Aggregation half times were fitted to a power law / A shows the same aggregation mechanism with and without Zn 2+ ions Abelein, Gräslund, Danielsson, 215, PNAS, in press Microscopic rate constants determine nucleation reactions Dependence of on nucleation reaction: Cohen et al., J Chem Phys (211), 135:6515 Cohen et al., J Mol Biol (212), 421, 16-171 Meisl et al., PNAS (214), 111, 9384-9389
Global fit analysis of kinetic traces Time dependence of fibril mass fraction: with global fit parameter for primary nucleation and secondary nucleation Meisl et al., PNAS (214), 111, 9384-9389 Effect of Zn 2+ on A aggregation Inhibition of A aggregation exhibits an exponential dependence on [Zn 2+ ] Experiments without and with preseeding follow the same trend. Zn 2+ primarily affects fibrilend elongation Coloured: not pre-seeded; black: pre-seeded
Molecular mechanism of A -Zn 2+ interaction 1 H- 15 N HSQC Translational diffusion: Zn 2+ binds to the N-terminus and forms a compact complex NMR relaxation dispersion experiments magnetization evolution time 1/2 Hz 15 N 18 2n Carr-Purcell-Meiboom-Gill (CPMG) pulse train scheme Information from relaxation dispersion: - structural information: Δ - thermodynamic information: - dynamic information: of the system: of the molecule: from two global fit parameters, and, and two resdiue-specific fit parameters, Δ and Palmer III et al., Methods Enyzmol., 21, 339, 24-238 Mittermaier & Kay, Trends in Biochem Science, 29, 34, 61-611 Hansen et al., J Biomol NMR, 28, 41, 113-12
Chemical exchange and thermodynamics E3 Thermodynamic binding features: [1] The Zn 2+ -A complex is highly dynamic and only marginally stable Model of Zn(II) effect on Aβ fibril formation
Acknowledgement Cooperations: Axel Abelein, Stockholm University Jens Danielsson, Stockholm University