Simulations of Peptide Aggregation

Joan-Emma Shea

Department of Chemistry and BiochemistryThe University of California, Santa Barbara

Protein and Peptide Aggregation

PROTEIN AGGREGATION AND DISEASE

Alzheimer

Huntington

Parkinson

Prion(“Mad Cow”)

Proteins not associated with a specific disease can also aggregate to form amyloid fibrils

APPLICATION TO BIOMATERIALS

Nano-tube and nano-sphere fabrication using aromatic di-peptides. [Gazit et al., Nano Lett, 4 (2004) 581]

Use of peptides to form nanoscale-ordered monolayers, COSB 2004, 14: 480

Time scalesSide-chain rotations

Loop closure

Helix formation Folding of-hairpins

Protein folding

Protein aggregation

All-atom: MolecularDynamics (MD)With EXPLICIT Solvent

MULTISCALE APPROACH

                         

                         

Off-lattice minimalist:Langevin dynamics

All-atom: MolecularDynamics (MD) withIMPLICITsolvent

COARSE GRAINING

1. Dimerization Mechanisms of 4 tetrapeptides KXXE

2. Design of Inhibitors of aggregation of the AlzheimerAmyloid-beta (A) peptide.

Fibrilsof KFFE

KXXE peptides

Tjernberg et al. , JBC 277 (2002) 43243

KAAE

KVVE KFFE

KLLE

Fibrils!

PEPTIDE MODEL

CHARMM19 Force Field and Generalized Born Implicit Solvent

1010

54)(

RrRrr

RerU pc

R

ep=1 kCal/mol, =1Å,

R=17Å

Confining Sphere:

NVTNVTT1

TN

T2

))((,1min mnij EEe

Simulation protocol: Replica Exchange MD6 replicas for monomer: total simulation time: 40 ns12 replica for dimers: total simulation time: 400 ns

Heterogeneous dimers

RMSD (Å)

E (

kC

al/m

ol)

Association temperature Ta

KFFE > KVVE > KLLE > KAAE

Thermodynamicstability of dimers:

Aa

La

Va

Fa TTTT

What determines transition temperature Ta?

Ta=

SU / translconfig SS

Propensity for structureSalt bridgesHydrophobic contactsAromatic interactions

monomererchain UU int

T=325K

T=240K T=275K

Free energy as a function of interaction energy and radius of gyration

T=285K

U (kCal/mol)

Rg

(Å

)

U

UU

T=325K

T=240K T=275K

Free energy as a function of interaction energy and radius of gyration

T=285K

U (kCal/mol)

Rg

(Å

)

U

UU

Interaction energy: KFFE > KLLE ~ KVVE > KAAE

Most favorable

Least favorable

Entropy loss due to dimerization

Monomers, Rg>5A

-strand

RandomCoil

Helix

Entropy loss due to dimerization

Monomers, Rg>5A

-strand

RandomCoil

Helix

Entropy loss due to dimerization

Monomers, Rg>5A Dimers, Rg<5A

-strand

RandomCoil

Helix

Entropy loss due to dimerization

Monomers, Rg>5A Dimers, Rg<5A

-strand

RandomCoil

Helix

-strand basin more populated for monomerof KVVE than KLLE: SL > SV

Association temperature

SUTa /

Energetic effect

Entropic effect

FVAL SSSS

ALVF UUUU ~

Aa

La

Va

Fa TTTT

KFFE KLLE

Kinetic accessibility of dimers

E (Kcal/mol) E (Kcal/mol)

Rg

(Å

)

KFFE KLLE

“DOWNHILL” DIMERIZATION FOR KFFE

FREE ENERGY BARRIERFOR KLLE

Different mechanisms of dimerization

PHE-PHE come together first (stabilized by vdw interactions), followed by the overall collapse of the structure with formation of peptide backbones contacts .

Rg

ove

r C

Rg over PHE atoms Rg over LEU atoms

LEU side chain formation and overall collapse with formation ofpeptide backbone interactions occur simultaneously.

KFFE KLLE

CONCLUSIONS

Aa

La

Va

Fa TTTT for dimerization of the

KXXE peptides1.

2. Strong sequence dependence of free energy landscapes for dimerization, with KFFE experiencing a barrierless transition.

3. KFFE dimers are the most thermodynamically stable and kinetically accessible.

4. Dimer trends match experimental trends observed for fibrils.

Aβ40: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVV

Aβ42: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVVIA

LARGER SYSTEMS: OLIGOMERIZATION OF THE ALZHEIMER AMYLOID BETA (A) PEPTIDES

AMYLOID BETA (A) PEPTIDES AGGREGATE TO FORM TOXIC OLIGOMERS AND FIBRILS

Aβ40: DAEFRHDSGYEVHHQKLVFFAEDV25GSNKGAIIGL35MVGGVV

FRAGMENT 25-35 OF THE (A) PEPTIDE APPEARS TO BETOXIC IN MONOMERIC, SMALL OLIGOMERIC AND FIBRILLAR FORMS

NO STRUCTURE OF THE MONOMERIC PEPTIDE AVAILABLE IN AQUEOUS SOLVENT

PEPTIDE ADOPTS A HELICAL STRUCTURE IN APOLARORGANIC SOLVENT (SUCH AS HEXAFLUOROISOPROPANOL HFIP)

EFFECTS OF SOLVENT ON FREE ENERGY LANDSCAPE OF THE MONOMER

REPLICA EXCHANGE SIMULATION IN:

1) HFIP/WATER CO-SOLVENT 2) PURE WATER

GROMOS96 FORCE FIELD, EXPLICIT SOLVENT

40 REPLICAS, 16 NS EACH, TOTAL SIMULATION TIME OF 640 NS

FREE ENERGY SURFACE IN HFIP/WATER COSOLVENT:

HELIX STABILIZATION

T=300 K

45 %

8%

N

N

N

N

C

CC

C

HFIP DISPLACES WATER NEAR THE PEPTIDE, AND FORMS A “COAT” AROUND THE PEPTIDE

FREE ENERGY SURFACE IN PURE WATER

FORMATION OF COLLAPSED-COILS and -HAIRPINS

T=300K

N

N

N

N

N C

C

C

C

C

Possible importance of turn in toxicity of monomer

REPLICA EXCHANGE MD ON DIMERS IN WATER:HETEROGENEOUS DIMERS

TWO TYPES OF ORDERED DIMERS

Experimentally: two different protofilament of diameters:1.41 +/- 0.48 nm3.58 +/- 1.53 nm

PEPTIDE INHIBITORS OF ALZHEIMER A AGGREGATION

Alzheimer’s disease (AD) is a neurodegenerative disease of the central nervous system.

ALZHEIMER DISEASE IS CHARACTERIZED BY THEPRESENCE OF NEUROFIBRILLAR TANGLES AND

AMYLOID PLAQUES IN THE BRAIN

Aβ40: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVV

Aβ42: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVVIA

AMYLOID PLAQUES CONSIST OF AMYLOID BETA (A) PEPTIDES GENERATED FROM THE PROTEOLYTIC CLEAVAGE OF THE APP TRANSMEMBRANE PROTEIN

Amyloid Plaques

Aβ40: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVV

Aβ42: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVVIA

In healthy individuals, the A peptides are broken down and eliminated. In AD, these peptides self-assemble into amyloid fibrils

Fibril

Both small soluble oligomers and fibrils appear to be toxic to cells.

Native Protein

Misfolded Protein

Soluble Oligomer

Protofibrils

Fibril

N-methylated A(16-20)m peptides can:1) prevent the aggregation of full length A peptide 2) disassemble existing fibrils and possibly small oligomers.

Aβ40: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVV

N-METHYLATED PEPTIDE INHIBITORS

16K(me)LV(me)FF

Fibrils ofA(1-40)peptides

After IncubationwithA(16-20)mpeptides

Meredith and co-workers, J. Pep. Res. (2002) 60, 37-55

Fragment A(16-22) KLVFFAE aggregates to form fibrils

Tycko et al. Biochemistry, 39 (45), 13748 -13759, 2000

Antiparallel arrangements from solid state NMR

MODEL SYSTEM

Aβ40: DAEFRHDSGYEVHHQ16KLVFFA22EDVGSNKGAIIGLMVGGVV

A(16-22) KLVFFAE PROTOFIBRIL

INITIAL STRUCTURE:

Two parallel bilayers

Peptides in layer antiparallel

lys16 and glu22 point to solvent

leu17, phe19, ala21 point inside core

A(16-22) KLVFFAE PROTOFIBRIL

INITIAL STRUCTURE:

Two parallel bilayers Peptides in layer antiparallel

lys16 and glu22 point to solvent

leu17, phe19, ala21 point inside core

GROMOS96 FORCE FIELDEXPLICIT SPC WATER(23000 atoms)

REACTION FIELD/ PME TWO 20 NS SIMULATIONS

REPRESENTATIVE A(16-22) PROTOFIBRIL

(LAST 7 NS OF SIMULATIONS)

Distance between bilayers: 0.93 nm (Tycko: 0.99nm)Distance between peptides: 0.44-0.52 nm (Tycko: 0.47 nm)

Structure of N-methylated A(16-20)m Inhibitor Peptide

Replica Exchange Molecular Dynamics, 30 replicas,20 ns per replica

A(16-20)m more rigid than A(16-22), with -strand content:This pre-organization may allow A(16-20)m to successfullycompete with free A(16-22) for binding to fibril.

Interaction of A(16-20)m Inhibitor Peptide with protofibril

INITIAL STRUCTURE

A(16-20)m with N-methyl groups pointing away fibril

A(16-20)m with N-methyl groups pointing toward fibril

Interaction of A(16-20)m Inhibitor Peptide with protofibril

AFTER 50 ns

A(16-20)m with N-methyl groups pointing toward fibril

Intercalates betweenlayers

A(16-20)m with N-methyl groups pointing away fibril

Forms hydrogen bonds with fibril(antiparallel)

POSSIBLE MECHANISM OF FIBRIL DISRUPTION

Inhibitor drifts from edge of fibril to side and inserts in fibril (between strands 6 and 7) with Lys pointing to solvent and hydrophobic residues inserted in fibril.

POSSIBLE MECHANISM OF INHIBITION OF FIBRIL GROWTH

Inhibitor forms hydrogen bond with fibril, with antiparallelalignment, possibly preventing additional A(16-22) peptidesfrom binding.

DESTABILIZATION OF FIBRIL WHEN INHIBITOR INSERTEDIN FIBRIL

Inhibitor inserted in fibril affects the “twist” of the fibriland the distance between strands.

CONCLUSIONS AND FUTURE DIRECTIONS

1. Results suggest possible mechanisms of fibril inhibition and disassembly

2. Extend the simulations to consider other orientations of the inhibitor peptides

3. Study and design new inhibitors

ACKNOWLEDGEMENTS

Dr. P. Soto

Other group members: M. Friedel, M. Griffin, A. Jewett, W. B. Lee and E. Zhuang

Funding: NSF Career, David and Lucile Packard Foundation, A. P. Sloan Foundation, Army Research Office.

Collaborator: Prof. Stephen Meredith, University of Chicago

Dr. G. WeiDr. A. Baumketner

University of California Santa Barbara