Copper and Zinc Copper and Zinc Coordination mode in Coordination mode in --

Amyloid PeptidesAmyloid Peptides

A XAS and A XAS and ab initio ab initio studystudy

ABR2008, 10-11 Aprile 2008, RomaABR2008, 10-11 Aprile 2008, Roma

V. MinicozziV. MinicozziPhys. Dept.- University of RomePhys. Dept.- University of Rome

Tor VergataTor Vergata

Calculations

DESY -BerlinK. JansenN. Christian

Experiments

CNR- TrentoM. Dalla SerraC. Potrich

EMBL-DESY-HamburgW. Meyer-Glauche

Biophysics Group in Rome

“Tor Vergata”

S. MoranteG. C. RossiV. MinicozziF. StellatoS. Alleva

A. Maiorana

Computations have been performed at

Fermi

BEN

Altix

SummaSummaryry

• A peptide

• XAS results

• ab-initio simulations

• Conclusions and outlook

Alzheimer Disease (AD)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

Amyloid Amyloid -peptide-peptide

- & -secretases cleavage non-pathological peptide P3

- & -secretases cleavage pathological peptides A1-40, A1-42

• A is derived from proteolitic cleavage of APP protein (Amyloid Precursor Protein).

•APP: 770 trans-membrane protein coded in chromosome 21

APP

APP

P3

17 40-42

A

1 40-42

-secretase

-secretase

-secretase

Cu2+

EPRA.K. Tickler et al. (2005) JBC 280:13355

Cu2+

NMRJ. Danielsson et al. (2007) FEBS 274:46

Zn2+

NMR

Syme & Viles (2006) BBA 1764:246S. Zirah et al. (2006) JBC 281:2151

NMR

Zn2+

DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV

Cu2+/Zn2+- A1-16

Cu2+/Zn2+- A17-40

Cu2+/Zn2+- A1-28

Cu2+/Zn2+- A5-23

minimal fragment containing His6, His13, and His14, 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 Experiments on…on…

EXAFSEXAFS

Cu2+/Zn2+-A17-40 = Cu2+/Zn2+- buffer

Cu2+-A1-16 = Cu2+- A1-28 = Cu2+-A1-40

Cu2+-A5-23 ≠ Cu2+-A1-16

Zn2+-A1-16 = Zn2+-A1-28 = Zn2+-A5-23 = Zn2+-A1-40

-8

-4

0

4

4 6 8 10

Zn-A

Zn-A

Zn-A

Zn-A

Zn-A

k(Å) -1

-8

-4

0

4

4 6 8 10

Cu-A

Cu-A

Cu-A

Cu-A

Cu-A

k(Å) -1

•3 Histidines•1 Tyrosine•1 O

Cu-A1-16EXAFSEXAFS

0 1 2 3 4 5

DataFit

|FT|

r(Å)

-8

-4

0

4

4 6 8 10

DataFit

Cu-A1-16

k(Å-1)

•2 Histidines•1 N-term•1 Tyrosine•1 O

Cu-A5-23EXAFSEXAFS

-8

-4

0

4

4 6 8 10

DataFit

k(Å-1) 0 1 2 3 4 5

DataFit

|FT|

r(Å)

•4 Histidines•1 O

Zn-A1-16EXAFSEXAFS

-8

-4

0

4

4 6 8 10

DataFit

Zn-A1-16

k(Å-1) 0 1 2 3 4 5

DataFit

r(Å)

|FT|

• Metal binding site lies within the first 1616 aminoacids

• Cu2+ and Zn2+ have different binding geometry

• Zn2+-A less rigid geometry, sensitive to solution condition

• Cu2+-A very stable binding mode

• Zn2+-A inter-molecular binding suggests aggregation

• Cu2+-A intra-molecular binding

XAS Conclusions

Questions: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

parallel version of Quantum-ESPRESSO package ⇨ CP-MD⇨ CP-MD

CP method is based on DFT theory

• Vanderbilt ultrasoft pseudopotentials • Perdew-Burke-Ernzerhof (PBE) exchange-correlation (xc) functional

ℒ ij

ijjiijIiI

IIi

i EM ][,2

1 22 RRr

Enforcing the orthonormality of KS wave functions

Nuclei move experiencing both the force due to electrons, , and the force due to electrostatic nuclear interaction,

DFTE

NE

Fictitious dynamics for electrons ⇨ ⇨ electronic degrees of freedom i

j

jiji

iE

A-peptide systems

Classical MD simulations of CuClassical MD simulations of Cu+2+2-A-A1-161-16 in water: in water:

- Cu+2 bounded to His6, Tyr10, His13, His14

- Cu+2 bounded to Nterm, His6, His13, His14

Classical MD simulations of ZnClassical MD simulations of Zn+2+2-A-A1-161-16 in water: in water:

- Zn+2 bounded to His6, Tyr10, His13, His14

- Zn+2 bounded to 4 Histidines 2 A1-16

CP-MD simulations of Cu complexesCP-MD simulations of Cu complexes

S1 Cu+2(D1-2-E3-4-5-H6-cap cap-H13-H14-cap) + 125 H2O

S2 Cu+2(D1-2-3-4-5-H6-7-8-9-Y10-E11-12-H13-H14-cap) + 180 H2O

S3 Cu+2(D1-2-3-4-5-H6-7-8-9-Y10-E11-12-H13-H14-cap) + 158 H2O

S2 Cu+2(D1-2-3-4-5-H6-7-8-9-Y10-E11-12-H13-H14-cap) + 180 H2O

comes from classical MD of Cu+2-A1-16 with Cu+2 bound to His6, Tyr10, His13, His14

S3 Cu+2(D1-2-3-4-5-H6-7-8-9-Y10-E11-12-H13-H14-cap) + 158 H2O

comes from classical MD of Cu+2-A1-16 with Cu+2 bound to Nterm, His6, His13, His14

S1 494 atoms and 13691369 electronsS2 703 atoms and 1951 electronsS3 628 atoms and 1776 electrons

S1: Distances from Cu2+

S2: Distances from Cu2+

Conclusions and OutlookConclusions 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

Thanks for your Thanks for your attention!attention!

XANESXANES

Cu2+-Aβ1-16 = Cu2+-Aβ1-28 = Cu2+-Aβ1-40 ≠ Cu2+-Aβ5-23 ≠ Cu2+-Aβ17-40

Zn2+-Aβ1-16 = Zn2+-Aβ1-28 ≠ Zn2+-Aβ1-40 ≠ Zn2+-Aβ5-23 ≠ Zn2+-

Aβ17-40

Feasibility studies for S1 systemFeasibility studies for S1 system

Platform CPU hours for 104

steps

Fermi1 in a 16 node configuration

1650

BEN in a 16 node configuration 1300

Altix in a 16 node configuration 300

- 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 S1Altix scaling on S1

V.Minicozzi, et al. (2008)  International Journal of Quantum Chemistry in press

S1: Dihedral angle Nδ(H6)–Cu–N(H13)–Nδ(H14) as a function of the CP simulation

time