Structural studies of amyloidogenic peptides and proteins Annette - - PowerPoint PPT Presentation

Structural studies of amyloidogenic peptides and proteins

Annette Eva Langkilde

• Dept. Of Drug Design and Pharmacology / Dept. Of Chemistry

University of Copenhagen, Denmark

• Disease Related
• Unbranched
• Extracellular
• In vivo
• Green birefringance upon Congo Red binding
• Cross-β fiber diffraction pattern
• … and lots of amyloid-like fibrils

Amyloid fibrils

www.alzheimersinfo.info

• Am. Soc.Hematology
• E. coli Biofilm AJC1/Flickr

pdb-code 1d0r, GLP1

Fibril Structure

• Jimenez et al, 2002, PNAS

Human Insulin GNNQQNY fibrils Nelson et al, 2005, Nature Peptide/yeast prion protein Fitzpatrick et al, 2013, PNAS TTR 105-115

aSN

• A. van Maarschalkerweerd

The fibrillation process

Lag-phase (nucleation) Stationary phase (maturation) Growth phase (elongation)

Nelson et al, 2005, Nature Peptide/yeast prion protein Lashuel et al. JMB a-synuclein Langkilde, Vestergaard Peptide/yeast prion protein Hua & Weiss (2004) Insulin, pH~1 Losic et al. 2006, J.

• Struct. Biol Aβ(1-40)

Jimenez et al, 2002, PNAS Human Insulin Grønning, Vestergaard Human Insulin

Method? SAXS!

fibril Fibrillation prone conformation Nucleus? On-pathway

• ligomer

Fibrillation related off- pathway

• ligomer

Amorphous aggregate Native unfolded monomer Native interactions Partially (un)folded (membrane vicinity)

?

Amorphous aggregate FIBRIL strain 1 FIBRIL strain 2 Alternative fibrillation pathways Native folded monomer Native oligomer

1000 2000 3000 4000 5000 6000 5 10 15 20

ThT-emission [rfu] Time [h]

y = (m1)+((m3)/(1+exp(-(m0-m... Error Value 207.13

• 119.03

m1 262.24 4315.1 m3 0.34187 8.1073 m5 0.30351 1.5765 m6 NA 9.4279e+6 Chisq NA 0.96673 R

Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251 Langkilde AE, Vestergaard B. (2012) Meth.Mol. Biol. 849, 137-55

Time resolved SAXS during fibrillation (α-synuclein)

SAXS data – additive

nk: volume fraction Ik(q): scattering intensity from the k-th type of particle K: number of components

*

How many species? Normalized eigenvectors

Momentum transfer Component Log(eigenvalue)

3 components: Itot=xIa+yIb+zIc

Singular Value Decomposition (SVDplot)

3 components: Itot=xIa+yIb+zIc

Isolating the scattering curves - OLIGOMER

3 components: Itot=xInative+yI???+zIfibril Itot=xInative+yIunknown+zIfibril

Get x, y and z estimates Recalculate and refine using residuals

1000 2000 3000 4000 5000 6000 5 10 15 20

ThT-emission [rfu] Time [h]

• 119.03

Itot=xInative+zIfibril

Get residuals, as first estimate

Decomposition of species (α-synuclein)

Fibril Monomer/dimer Oligomer ThT

Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

Compare typical distances

Mature fibril Mature fibril cross-section Oligomer

Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

Corresponding sizes… Do the oligomers build the fibrils? Ab initio models of αSynuclein fibrillar species

180Å Giehm L, Svergun DI, Otzen DE, Vestergaard B (2011) PNAS 108, 3246-3251

When it doesn’t work….

αSN E46K

Itot=xInative+yIunknown+zIfibril

Get x, y and z estimates Recalculate and refine using residuals

Itot=xInative+zIfibril

Get residuals, as first estimate

• A. Van Maarschalkerweert, PhD thesis, 2014

αSN E46K

• A. Van Maarschalkerweert, PhD thesis, 2014

Development of an OBJECTIVE ROUTINE for the decomposition

Chemometric method: Multivariate Curve Resolution using Alternating Least Squares (MCR-ALS) Fátima Herranz-Trillo & Prof. Pau Bernado, U. Montpellier;

• Prof. Roma Tauler, U. Barcelona Bente Vestergaard, U. Copenhagen

Reduce Ambiguities: Use of multiple data matrices Absolute scale (I Vs s) Kratky plot (I·s2 Vs s) Porod plot (I·s4 Vs s) Holtzer plot (I·s Vs s)

χ2〉 = 1.16 χ2〉 = 1.04 χ2〉 = 1.03 χ2 = 3.98 χ2 = 2.08

1240 monomers 1 monomer

Insulin fibrils

~ 5-6 monomers

Vestergaard B, Groenning M, Roessle M, Kastrup JS, van de Weert M, Flink JM, Frokjaer S, Gajhede M, Svergun DI (2007) PLoS Biol, 5, e134

Insulin & E46 revisited

Coming soon to a journal near you… Structural Analysis of Multicomponent Amyloid Systems by Chemometric SAXS Data Decomposition

Herranz-Trillo F, Groenning M, van Maarschalkerweerd A, Tauler R, Vestergaard B and Bernadó P Structure (2016) Accepted

fibril Fibrillation prone conformation Nucleus? On-pathway

• ligomer

Fibrillation related off- pathway

• ligomer

Amorphous aggregate Native unfolded monomer Native interactions Partially (un)folded (membrane vicinity)

?

Amorphous aggregate FIBRIL strain 1 FIBRIL strain 2 Alternative fibrillation pathways Native folded monomer Native

• ligomer

Nelson et al (2005) Nature, 435, 774-449

Full-length proteins vs peptide fragments

GNNQQNY

Langkilde, Morris, Serpell, Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882–895

GNNQQNY

• No lag-phase
• No oligomer build-up
• Monomeric starting state
• Ribbon like fibrils
• Bragg peak (late fibril state)

Fibril cross-section P(r)

Fibre diffraction

Method and sample prep: Morris, K. L. & Serpell, L. C (2012) Meth.Mol. Biol. 849, 121-35.

SAXS vs FD - complementarity

SAXS data recorded at ID14-EH3, ESRF Detector distance: 2.34 m Wavelength: 0.931 Å Resolution range :~ 1000 - 10 Å FD recorded at 911:2, MAXlab Detector distance: 0.22 m Wavelength: 1.04 Å Resolution range :~ 50 - 3 Å

• A. E. Langkilde

Small-angle X-ray scattering (in solution)

SAXS data recorded at ID14-EH3, ESRF Detector distance: 2.34 m Wavelength: 0.931 Å Resolution range :~ 1000 - 10 Å

Log I q (s) nm-1

• A. E. Langkilde

Combining SAXS, FD, and TEM

c=48 Å b=4.7 Å a=31 Å

48 Å

Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882–895

Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882–895

Packing model

Fiber diffraction simulation - CLEARER

c a

Simulated diffraction image Experimental diffraction image

Langkilde, Morris, Serpell. Svergun, & Vestergaard, Acta Cryst. (2015). D71, 882–895

Transthyretin (TTR)

• Native state: 55 kDa Homotetramer
• TTR misfolding and aggregation  amyloid diseases
• wildtype ATTR amyloidosis (senile systemic amyloidosis)
• familial amyloid polyneuropathy
• familial amyloid cardiomyopathy

Protofibrils/ Fibrils Native tetramer Native monomer Misfolded monomer

TTR dH2O ___ TTR 3.5 min ….. TTR 10 min TTR unfolded (11% ac.ac.)

SAXS on TTR fibrillation

• Pre-ini MW ≈ 3.3 monomers
• Pre-ini ≠ xtal
• Pre-ini ≠ xtal+dimer/monomer
• Pre-ini = 85% xtal + 15% unfolded

~2900 kDa ~215 protomers

Groenning et al. & Vestergaard (2015) Scientific Reports

TTR toxicity

The unfolded state

*

Some practical aspects

• Test your system, find optimal conditions
• Know your system
• complementary methods, e.g. TEM and FD (”SAXS and …” talks tomorrow)
• Consider beamline stability, time frames, additional equipment etc
• Check 2D images
• Check buffers, basic parameters
• ...and double check!
• Test different inputs and parameters (consistent solutions)

Langkilde AE, Vestergaard B (2012) Methods Mol Bio, 849, 137-155

Acknowledgements

University of Copenhagen

• Bente Vestergaard
• Minna Grønning
• Andreas van Maarschalkerweerd

EMBL, Hamburg DE

• Dmitri Svergun,
• Manfred Rössle
• BioSAXS group

Aarhus University, DK

• Lise Giehm
• Danial Otzen

Univesity of Sussex, UK

• Louise Serpell
• Kyle Morris

Funding The Lundbeck Foundation Carlsberg Foundation The Danish Council for Independent Research | Medical Sciences CoNEXT – University of Copenhagen Danscatt BiostructX Beamlines and local contacts DESY/PETRAIII, Hamburg, DE X33, P12 ESRF, Grenoble, FR ID14-3, BM29 MAX-lab, Lund, SE 911:2, 911:3 CBS, Montpellier, FR

• Fatima Herranz-Trillo
• Pau Bernado

IDAEA-CSIC, Barcelona, ES

• Roma Tauler

Linköping University, SE

• Per Hammarström
• Raul I Campos
• Daniel Hirschberg