Human Prion Protein Amyloid:A Tale of Structure and Stability

Nathan J. Cobb

Department of Physiology and Biophysics

Case Western Reserve University, Cleveland, OH

The Prion Diseases

Disease Pathogenesis

Human Diseases• Creutzfeldt-Jakobs disease (CJD) Sporadic, iatrogenic

• variant CJD Infection from bovine prions?

• Familial CJDs Inherited

• Kuru Infection

• Gerstmann-Straussler-Sheinker (GSS) Inherited

• Fatal familial insomnia Sporadic, Inherited

Animal Diseases• Scrapie (sheep) Sporadic, Infection

• Bovine spongiform encephalopathy Sporadic, Infection

• Transmissible mink encephalopathy Sporadic, Infection

• Chronic wasting disease (cervids) Sporadic, Infection

• Feline spongiform encephalopathy Infection

• The infectious agent in the transmissible spongiform encephalopathies

(TSEs) is not a virus, but a misfolded form of the normal (cellular) prion

protein, PrPC

• This misfolded form, PrPSc (scrapie), is a β-sheet-rich aggregate which arises

from conformational conversion of the normally monomeric and primarily

α-helical PrPC

• Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s,

TSEs are often associated with neuronal accumulation of amyloid deposits

• PrPSc self-perpetuates by first binding PrPC and then inducing conformational

conversion of the latter protein to the PrPSc state

• Although the ‘Protein-only’ nature of the TSEs has yet to be conclusively

shown, studies in yeast and other fungi have provided the proof-of-principle

that proteins alone can act as self-perpetuating agents

• Challenge to the ‘Anfinsen Principle’?

The ‘Protein-Only’ Hypothesis of Prion Diseases

Leader Sequence: 1-22

Octapeptide Repeats: 51-91

-Helicies: 144-156,174-194, 200-228

-Sheets: 128-131, 161-164

Disulfide Bond: 179 and 214

N-linked Glycosylation: 181, 197

GPI Anchor: 231

GPI Anchor Signal: 232-254

The Structure of Cellular Prion Protein

S S

G

H1

1H2 H3

G

G

P

IPHGGGWGQ

PrPC PrPSc

23

S S

Proteinase K

S S

Proteinase K

S S

231

231~87-90

23 231

Digestion with Proteinase K

Surrogates for Structural Information

Congo Red

Thioflavin T

Binding of ‘Amyloid-Specific’ Dyes

Immunostaining for PrP

Immunostaining for Glial

Fibrillary Acidic Protein

Staining with

Hematoxylin-Eosin

Aguzzi et al. (2001) Nature

-39 kDa

-28 kDa

-14 kDa

-19 kDa

PrPC PrPSc

PK - + - +

FTIR spectra of brain-derived PrP

— PrPC

- - - PrPSc

••••• PrP 27-30

Pan et al. (1993) PNAS

Amyloid Fibrils

Nelson et al. (2005) Nature

X-ray diffraction structure of microcrystals

formed by the peptide GNNQQNY

Petkova et al. (2004) PNAS

Aβ1-40 structure as determined

by solid-state NMR

Like other neurodegenerative diseases such as Alzheimer’s and Parkinson’s,

TSEs are associated with neuronal accumulation of amyloid deposits

0 5 10

0.0

0.5

1.0

1.5

2.0

2.5

Th

T f

luo

res

ce

nc

e

Time, hrs

2% seed

No seed

Conversion of rPrP (residues 90-231) into Amyloid Fibrils: ‘Synthetic Prions’

Nucleation

Elongation

Fragmentation 2o Nucleation

What is the Relevance of rPrP Amyloid to PrPSc?

• It has been shown that in vitro converted amyloid fibrils induce a TSE-like

disease in mice overexpressing PrP89-231 (Legname et al. (2004) Science)

• The infectivity titer is very low, suggesting that only a small fraction of this

material is infectious

• rPrP amyloid allows for structural and biochemical characterization techniques

that are not amenable to brain-derived aggregates

Brain-Derived PrPSc ‘Synthetic Prions’

Glycosylated Unglycosylated

Membrane-associated NA

16 kDa PK-resistant core Shorter PK-resistant core

Site-Directed Spin Labeling

Nitroxide-labeled proteins yield three important pieces of information

Involves substitution of cysteine for

native residues followed by thiol-specific

modification with the nitroxide reagent

1) Distance estimates – dipolar broadening and spin exchange

2) Mobility information

3) Accessibility

EPR spectroscopy

Absorption of electromagnetic radiation by an unpaired electron

in an applied magnetic field

H

En

erg

y

MS = 1/2

MI = 1/2

Hres

signal

amplitude

modulation amplitude

MI = -1/2

MS = -1/2

H

Energ

y

H

Absorp

tion

Hres

H

Absorp

tion

H

D(A

bsorp

tion

)/dH

Hres

H

d(A

bsorp

tion

)/d

H

Nitroxide EPR spectra

H

+1/2

-1/2

En

erg

y

+1

0

-1

-1

0

+1

MI MS

Mobility

Margittai & Langen (2004) PNAS

Jayasinghe & Langen (2004) J. Biol. Chem

Spin Exchange

Distance Estimates

Dipolar Broadening

Initial Purification and Refolding of rPrP

Denaturing Wash (10 mM reduced glutathione, 6 M GdnHCl, pH 8.0)

Gradient from 6 M → 0 M GdnHCl

Wash

Low Imidazole (50 mM imidazole, pH 8.0)

Collect High Imidazole (350 - 500 mM imidazole, pH 5.8 - 6.4)

Standard Buffer: 100 mM phosphate, 10 mM Tris, pH 7.0

Final Purification and Labeling of rPrP

• Dialyze collected fractions into 50 mM actetate, pH 4.0

• Pool and concentrate, storage at -80 oC

• Incubate with 5x molar equivalents of TCEP at 4 oC, dilute with 50 mM phosphate,

pH ~7.0, and add 10x molar equivalents of nitroxide spin-label and thrombin

• Verify nitroxide-labeling and 6xHis-tag cleavage by MALDI

• Remove excess spin label, uncleaved rPrP, rPrP aggregates, and remaining

impurities by cation exchange chromatography

• Measure monomeric EPR signal, concentrate, aliquot and store protein at -80 oC

Characterization of rPrP Cysteine Variants

1) Verify native-like α-helical

content by CD spectroscopy

3) Verify native disulfide formation by HPLC/MS of tryptic digests

2) Verify the proper formation

of amyloid fibrils by AFM

Monomer

FibrilUndiluted Fibril

1:4 Spin-Diluted

Fibril

Spin Exchange in Labeled rPrP Amyloid Fibrils

EPR Signals for Nitroxide-Labeled rPrP Fibrils

Cobb et al. (2007) PNAS

Monomer

Fibril

4 M GdnHCl Fibril (no denaturant)

Fibril 4 M GdnHCl

Cobb et al. (2007) PNAS

Denaturation of Nitroxide-Labeled rPrP Fibrils

no denaturant

1 M GdnHCl

2 M GdnHCl

-helix -sheet1:4 dilution

undiluted

1:1 dilution

191 undiluted

190/191, 191/192

191 1:1 dilution

Only a Parallel In-Register -Structure can Describe EPR Data

Modeling of Prion Protein Amyloid

• Residues ~160-220 are in-register and parallel with one another

• The native disulfide is maintained in the amyloid structure

• Asn 181 and 197 must point toward the outside of the amyloid structure

PrP monomer

Two-bend modelOne-bend model

N181

N197

N181

N197

Molecular Details of the Two-Bend Model

N181

N197

Cobb et al. (2007) PNAS

Correlation with H/D Exchange Data

Lu et al. (2007) PNAS

23190

EPR data (160-220)

H/D exchange (170-220)

Pathogenic Mutations of the Prion Protein

2531 51 91

P102L

P105L

P105T

A117V

Y145stop

Q160stop

D178N

V180I

T183A

H187R

T188K

T188R

E196K

F198S

E200K

D202N

V203I

R208H

V210I

E211Q

Q212P

Q217R

M232R

P238S

23

Insertion of

1,2, or 4-9 repeats

EPR data (160-220)

H/D exchange (170-220)

Spiral model (116-164)

Left-handed helical model (89-175)

Left-handed

β-helical modelGovaerts et al.

(2004) PNAS

Spiral modelDeMarco and

Daggett (2004)

PNAS

Parallel an in-register

β-structure of rPrP amyloidCobb et al. (2007) PNAS

Structural Models of Prion Protein Aggregates

Sim and Caughey

(2008)

Neurobiol. Aging

Wille et al.

(2002) PNAS

90 231

Similar Amyloid Fibrils are Formed under Native Conditions

Native conditions 50 mM acetate

pH 4.0

Denaturing Conditions 50 mM phosphate

2M GdnHCl, pH 7.0

Cobb & Surewicz (2008) J. Biol. Chem.

pH-Induced Structural Differences at the N-Terminus are Reversible

Cobb & Surewicz (2008) J. Biol. Chem.

• Fibrils formed under

native (pH 4.0) conditions

• Equilibrated to pH 7.0

• Fibrils formed under denaturing

(2 M GdnHCl, pH 7.0)conditions

• Equilibrated to pH 4.0

193

103

132

193

103

132

113

113

103

192

Native and Denaturing Conditions Form rPrPFibrils of Similar Stability

Cobb & Surewicz (2008) J. Biol. Chem.

Monomer

Fibril

4 M GdnHCl

● Denaturing

○ Native

● EPR

Against GdnHCl Against pH

● Denaturing

○ Native

● EPR

Highly Acidic Conditions Stabilize rPrPAmyloid Fibrils

179

214

pH 4.0-10.0

179

214

pH 2.0

Fewer Stacked Charges

at Very Low pH

rPrP Amyloid is more Resistant

to GdnHCl at Low pH

Replacement of Hydrophobic with Charged Residues Yields Intriguing Results

Prion Strains and the Species Barrier

Cobb & Surewicz (2009) Biochemistry

23

S S

Proteinase K

S S

231

231~87-90

Structurally Distinct rPrP Amyloid Fibrils

4 M GdnHCl

2 M GdnHCl

500 nm

Conclusions

• As deduced by SDSL/EPR, the amyloid core of rPrP amyloid fibrils is comprised

of residues ~160/170-220 - there is no evidence of stable secondary structure

outside this β-core region

• The molecular architecture of rPrP amyloid fibrils is a parallel and in-register

stacking motif, where same residues are in direct contact with their counterparts

on neighboring molecules

• An identical amyloid core is formed under both denaturing and native conditions,

indicating that this region is strongly predisposed to forming amyloid fibrils

• Stacking of same charges appears to be the main destabilizing force in parallel

and in-register amyloid fibrils

• Substitution at only a single residue can result in structurally distinct amyloid

fibrils

• Varying buffer conditions can result in structurally distinct amyloid fibrils – the N-

terminal residues 23-90 are important for amyloid formation at high denaturant

concentrations