Characterizing -Synuclein Membrane Bound Structure

Bert Lai (Gray Group)Department of Chemistry and Chemical EngineeringCalifornia Institute of Technology

Thesis Defense04/29/08

Introduction: α-Synuclein • Role of α-syn in Parkinson’s disease pathogenesis unclear

• Aggregates form amyloid fibrils

• major component of Lewy Bodies deposits

• found in substantia nigra of Parkinson’s disease victims

• Three single point mutations

(A30P, A53T, and E46K)

• Early onset familial Parkinson’s

disease

Substantia nigra

Parkinson’s Disease

Normal

α-Synuclein Sequence

Three interesting regions:

1) Non β-Amyloid Component (NAC) region (Residues 61-95)

• Commonly found in Alzheimer’s disease aggregates

2) Acidic C-terminal region (Residues 96-140)

3) 7 imperfect repeats of 11 amino acids

• Second and seventh residues of each repeat are mostly KTKEGV

• Typically found in apolipoproteinsUlmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.

• Synaptic vesicle-associated protein

• Oligomers of α-syn involved in membrane permeabilization

• Structure:

• In solution: unfolded with structural preference

• In SDS micelles/acidic small unilamellar vesicles (SUVs):

highly helical

Introduction: α-Synuclein and Membrane

SDS micelles SUVs

1 nm20-50 nm

Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Biol. Chem. 2005, 280, 9595-9603.

NMR Model of α-Synuclein in Micelles

2nd Helix (Lys45 – Thr92)

1st Helix (Val3 – Val37)

Linker (Lys38 – Thr44)

Hydrophilic Tail (Gly93 – Ala140)

• Formation of α-helices at

• residue 3 to 37 (1st helix)

• residue 45 to 92 (2nd helix)

• Formation of linker between 2 helices

• Unstructured hydrophilic tail

Phospholipid Vesicles

• EPR: Helical structure in the 7 N-terminal 11-amino acid repeats

Jao, C. C.; Der-Sarkissian, A.; Chen, J.; Langen, R. Proc Natl. Acad. Sci. 2004, 101 (22), 8331-8336.

Lipid exposed

Solvent exposed

The CD spectra show that acidic lipid is needed for α-syn to form helical structures.

Phospholipids

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC)

1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphate (POPA)

1:1 POPC:POPA SUVs were made

For the N-terminus

Trp-Tyr(NO2) pairs

4

94

ro = 26 Å10 < r < 40 Å

W4-Y94

Helix #1

Helix #2

4

94

Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124 (19), 5481-5485.

FET Kinetics & Polypeptide Conformations

Steady State Fluorescence StudiesIn

tens

ity

W4 is in a more hydrophobic environment when associated with SDS or SUV

Wavelength (nm)

Amount of quenching by Y(NO2): SDS > SUV > Buffer

4

The two helices are closer together in SDS micelles. 94

4

NaPi

SDS

SUV

Distance (Å)

P(

r)

80 %

90 %

40 %

Time-resolved Fluorescence Studies

Time (ns)I t

SDS

SUV

Distance (Å)

P(

r)

Summary • Extended helix is elucidated when α-syn is in association with SUVs

• Similar to the EPR model

• α-syn forms compact structures when SDS micelles are introduced• Similar to the NMR model 94

4

Some extended structure remains

Calcium Binding Behavior of -Synuclein’s C-Terminal Tail

C-terminal Tail

Ulmer, T. S.; Bax, A.; Cole, N. B.; Nussbaum, R. L. J. Mol. Biol. 2005, 280, 9595-9603.

• locates in residues 96-140

• is unstructured in the presence of membrane mimic

• is highly negatively charged (10 Glus & 5 Asps)

• modulates aggregation

• binds to

• metal ions, e.g. calcium, copper

• microtubule-associated proteins 1B

• polyamines

Aging → Oxidative Stress → Calcium Dysregulation

Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.

de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.

Theory 1: C-terminal tail becomes β-sheet

Theory 2: C-terminal tail rigidified by binding calcium ion

Study C-terminal tail behavior in the presence of POPC:POPA with various Ca2+ concentration

Stage 1: Trp-only mutants

Stage 2: Donor-acceptor pair

DNP (N-Dinitrophenyl Phosphatidylethanolamine)

Br (6,7) POPC (1-Palmitoyl-2-Stearoyl(6,7-dibromo)-sn-Glycero-3-Phosphocholine)

McIntosh, T. J.; Holloway, P. W. Biochemistry 1987, 26, 1783-1788.

Quencher located on the end group

94 101

125 136

Headgroup/hydrocarbon boundary and quencher:3.5 Å

Trp Only Steady-state Fluorescence

NATA W125

W101 W136

Calcium Titration Curves (in SUVs)

[Ca2+] [Ca2+]

[Ca2+][Ca2+]

CD shows calcium ion does not affect the structure of α-synuclein.

CD

Time-resolved Fluorescent Lifetimes

W101 seems to be more associated with the membrane, but not deeply inserted.

W101 → DNP Fluorescence Transfer Rate

DNP + Ca2+

DNPA shift to shorter lifetimes is observed when calcium ions were added.

P(r

)

Log(k)

Tamamizu-Kato, S.; Kosaraju, M. G.; Kato, H.; Raussens, V.; Ruysschaert, J.-M.; Narayanaswami, V. Biochemistry 2006, 45, 10947-.

de Laureto, P. P.; Tosatto, L.; Frane, E.; Marin, O.; Uversky, V. N.; Fontana, A. Biochemistry 2006, 45, 38, 11523-.

Theory 1: C-terminal tail becomes β-sheet

Theory 2: C-terminal tail rigidified by binding calcium ion

80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

Acidic Residues

Mutation Sites

Ca Binding

Effective calcium binding has been shown for sequences DXXXD.

74

94101

125113

136

Seven mutants were expressed:

1) W94/Y113 6) W101/Y125

2) W94/Y125 7) W101/Y136

3) W94/Y136

4) W125/Y136

5) W101/Y74

Graphic made in PyMol

80Val Thr Gly Val Thr Ala Val Ala Gln Lys 90 Thr Val Glu Gly Ala Gly Ser Ile Ala Ala 94 100Ala Thr Gly Phe Val Lys Lys Asp Gln Leu101 110 Gly Lys Asn Glu Glu Gly Ala Pro Gln Glu 113 120Gly Ile Leu Glu Asp Met Pro Val Asp Pro 125 130Asp Asn Glu Ala Tyr Glu Met Pro Ser Glu 136 140Glu Gly Tyr Gln Asp Tyr Glu Pro Glu Ala

Acidic Residues

Mutation Sites

Ca Binding

Effective calcium binding has been shown for sequences DXXXD.

Binding sites for Calcium in solution is found between residue 101-113 & residue 125-136