1. Copper and Zinc Coordination mode in  -Amyloid Peptides A XAS and ab initio study ABR2008, 10-11 Aprile 2008, Roma V. Minicozzi Phys. Dept.- University of Rome Tor Vergata

2. Calculations DESY -Berlin K. Jansen N. Christian Experiments CNR- Trento M. Dalla Serra C. Potrich EMBL-DESY-Hamburg W. Meyer-Glauche Biophysics Group in Rome “Tor Vergata” S. Morante G. C. Rossi V. Minicozzi F. Stellato S. Alleva A. Maiorana Computations have been performed at Fermi BEN Altix

3. Summary A  peptide XAS results ab-initio simulations Conclusions and outlook

4. Alzheimer Disease (AD) AD brains show two lesions 1- Amyloid Plaques: Extracellular deposits of A  peptide almost spherical with a 10-100 mm diameter 2- Neurofibrillar Tangles: Intracellular abnormal elicoidal fibers mainly composed by tau protein These two lesions can occur independently of each other Amyloid Plaques Neuron Neurofibrillar Tangles

5. Amyloid  -peptide  - &  -secretases cleavage  non- pathological peptide P3  - &  -secretases cleavage  pathological peptides A  1-40, A  1-42 A  is derived from proteolitic cleavage of APP protein (Amyloid Precursor Protein). APP: 770 trans-membrane protein coded in chromosome 21 APP P3 17 40-42 A  1 40-42  -secretase  -secretase  -secretase

6. Cu 2+ EPR A.K. Tickler et al. (2005) JBC 280:13355 Cu 2+ NMR J. Danielsson et al. (2007) FEBS 274:46 Zn 2+ NMR Syme & Viles (2006) BBA 1764:246 S. Zirah et al. (2006) JBC 281:2151 NMR Zn 2+

7. DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV Cu 2+ /Zn 2+ - A  1-16 Cu 2+ /Zn 2+ - A  17-40 Cu 2+ /Zn 2+ - A  1-28 Cu 2+ /Zn 2+ - A  5-23 minimal fragment containing His 6, His 13, and His 14, suggested to be involved in metal binding complementary sequence where none of these His’s is present besides the presence of these three His’s, a long hydrophobic region believed to be relevant in the aggregation process the N-terminal region of the A  -peptide can play any role in the metal binding process? Stellato et al., Eur Biophys J (2006) 35: 340 Minicozzi et al., (2008) J Biol Chem in press Experiments on…

8. EXAFS Cu 2+ /Zn 2+ -A  17-40 = Cu 2+ /Zn 2+ - buffer Cu 2+ -A  1-16 = Cu 2+ - A  1-28 = Cu 2+ -A  1-40 Cu 2+ -A  5-23 ≠ Cu 2+ -A  1-16 Zn 2+ -A  1-16 = Zn 2+ -A  1-28 = Zn 2+ -A  5-23 = Zn 2+ -A  1-40

9. 3 Histidines 1 Tyrosine 1 O Cu-A  1-16 EXAFS

10. 2 Histidines 1 N-term 1 Tyrosine 1 O Cu-A  5-23 EXAFS

11. 4 Histidines 1 O Zn-A  1-16 EXAFS

12. 16 Metal binding site lies within the first 16 aminoacids Cu 2+ and Zn 2+ have different binding geometry Zn 2+ -A  less rigid geometry, sensitive to solution condition Cu 2+ -A  very stable binding mode Zn 2+ -A  inter-molecular binding suggests aggregation Cu 2+ -A  intra-molecular binding XAS Conclusions

13. Questions: 1.precise location of metal binding site along the sequence 2.different zinc and copper role in aggregation processes A promising tool is ab initio molecular dynamics Car-Parrinello Molecular Dynamics simulations (CP-MD) Classical MD  atoms move in the chosen force field ab-initio MD  electrons are active quantum mechanical DOF

14. ⇨ CP-MD parallel version of Quantum-ESPRESSO package ⇨ CP-MD CP method is based on DFT theory Vanderbilt ultrasoft pseudopotentials Perdew-Burke-Ernzerhof (PBE) exchange-correlation (xc) functional ℒ Enforcing the orthonormality of KS wave functions Nuclei move experiencing both the force due to electrons,, and the force due to electrostatic nuclear interaction, ⇨ Fictitious dynamics for electrons ⇨ electronic degrees of freedom

15. A  -peptide systems Classical MD simulations of Cu +2 -A  1-16 in water: - Cu +2 bounded to His 6, Tyr 10, His 13, His 14 - Cu +2 bounded to Nterm, His 6, His 13, His 14 Classical MD simulations of Zn +2 -A  1-16 in water: - Zn +2 bounded to His 6, Tyr 10, His 13, His 14 - Zn +2 bounded to 4 Histidines  2 A  1-16 CP-MD simulations of Cu complexes S1  Cu +2 (D 1 -2-E 3 -4-5-H 6 -cap cap-H 13 -H 14 -cap) + 125 H 2 O S2  Cu +2 (D 1 -2-3-4-5-H 6 -7-8-9-Y 10 -E 11 -12-H 13 -H 14 -cap) + 180 H 2 O S3  Cu +2 (D 1 -2-3-4-5-H 6 -7-8-9-Y 10 -E 11 -12-H 13 -H 14 -cap) + 158 H 2 O S2  Cu +2 (D 1 -2-3-4-5-H 6 -7-8-9-Y 10 -E 11 -12-H 13 -H 14 -cap) + 180 H 2 O comes from classical MD of Cu +2 -A  1-16 with Cu +2 bound to His 6, Tyr 10, His 13, His 14 S3  Cu +2 (D 1 -2-3-4-5-H 6 -7-8-9-Y 10 -E 11 -12-H 13 -H 14 -cap) + 158 H 2 O comes from classical MD of Cu +2 -A  1-16 with Cu +2 bound to Nterm, His 6, His 13, His 14

16. 1369 S1  494 atoms and 1369 electrons S2  703 atoms and 1951 electrons S3  628 atoms and 1776 electrons

17. S1: Distances from Cu 2+

18. S2: Distances from Cu 2+

19. Conclusions and Outlook We can discriminate via ab-initio simulations among the structural models extracted from XAS experiments QM/MM simulations of the whole hydrated A  peptide based on structural XAS data expecially relevant for Zn +2 structures where pairs of peptides are involved New XAS experiments with Aluminium and Zinc

20. Thanks for your attention!

21. XANES Cu 2+ -Aβ 1-16 = Cu 2+ -Aβ 1-28 = Cu 2+ -Aβ 1-40 ≠ Cu 2+ -Aβ 5-23 ≠ Cu 2+ -Aβ 17-40 Zn 2+ -Aβ 1-16 = Zn 2+ -Aβ 1-28 ≠ Zn 2+ -Aβ 1-40 ≠ Zn 2+ -Aβ 5-23 ≠ Zn 2+ -Aβ 17-40

22. Feasibility studies for S1 system PlatformCPU hours for 10 4 steps Fermi1 in a 16 node configuration1650 BEN in a 16 node configuration1300 Altix in a 16 node configuration300 - Fermi1 Linux-clusters (E. Fermi Institute, Rome - Italy) based on 1.7 GHz Pentium IV processors - BEN Linux-cluster (ECT ∗ Institute, Trento - Italy) based on Intel/Xeon- 2.8 GHz processors - ALTIX 4700 (LRZ, Munich - Germany) based on Intel Itanium2 Madison 9M 1.6 GHz processors 3.6 ps at 300 K 64 nodes ~ 1 month Altix scaling on S1 V.Minicozzi, et al. (2008) International Journal of Quantum Chemistry in press

23. S1: Dihedral angle N δ (H 6 )–Cu–N  (H 13 )–N δ (H 14 ) as a function of the CP simulation time