Structural%20Studies%20of%20Amyloid-like%20fibrils

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Structural Studies of Amyloid-like fibrils

• Eisenberg Lab
• Howard Hughes Medical Institute, UCLA-DOE
  Institute for Genomics and Proteomics, Box
  951570, UCLA, Los Angeles CA 90095-1570
• 20th April 2005

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Aggregation Diseases
Amyloid Diseases
Prions
Amyloid-like
Alzheimers Ab Psi Sup35 Huntington Huntingtin Ribonuclease A
Alzheimers and tauopathies Tau Ure2 Ure3 Parkinsons a-synuclein T7 Endonuclease I
Diabetes II Amylin CJD PrP ALS (Lou Gehrigs) Superoxide dismutase
Injection amyloidosis Insulin BSE (mad cow) PrP
Dialysis amyloidosis ?2-microglobulin
Senile amyloidosis Transthyretin
Hereditary cystatin C amyloid angiopathy Human Cystatin C
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Different Tertiary/Quaternary Structures
Insulin
Jimenez et al., Proc. Natl. Acad. Of Sci., 2002.
?2-Microglobulin
Ivanova et al., Proc. Natl. Acad. Of Sci., 2004
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Hallmarks of Amyloid
Long, Unbranched Fibrils
4.77Å
11.7Å
Bind Congo Red and yield an apple green
birefringence
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Structures of Amyloid Fibrils

• What are the structures of the various amyloid
  fibrils?
• Is the amyloid state a property of the main-chain
  interactions or is dependent on the side chain
  interactions as well?
• Does the formation of the amyloid require a
  complete refolding of the protein or does only a
  part of the protein contribute to the amyloid
  core while the rest of the protein essentially
  retains its native fold?

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Structures of the Cross-? Spines
Balbirnie at al., Proc Natl Acad Of Sci., 2001
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GNNQQNY forms Amyloid-like fibrils and Related
Microcrystals
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Christian Riekel
Anders Madsen
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Data collection at ESRF
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Microcrystal diffraction at ESRF
Scale Bar 10µm
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GNNQQNY Structure
Nelson et al., Nature 2005
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Hydrogen Bonding
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Interfaces Between Sheets
High Shape Complementarity
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Tau

• 335-441 amino acids
• Six isoforms
• The core of the Paired Helical Filaments (PHFs)
  determined by Pronase digestion is 9.5kDa-12kDa
  and corresponds to the C-terminal repeat regions
  (First shown by Aaron Klug and others in 1988,
  PNAS)
• Unstructured
• Binds microtubules.

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Important Hexapeptide Motifs in Tau
Von Bergen et al., 2005
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Rosetta Energy Predictions for Tau
Thompson et al., Proc. Natl Acad of Sci., 2006
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Fibrils
Von Bergen et al., 2001
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Structure of VQIVYK
Val1
Tyr5
Ile3
Val4
Lys6
Gln2
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Tyr5
Ile3
Val1
Val4
Lys6
Gln2
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Hydrogen Bonding
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Interfaces
Dry Interface
Wet Interface
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Hetero-Interfaces
Q
K
I
Y
V
I
Q
I
V
V
K
Q
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Complete Refolding Model

• Model is based on cryo-EM
• reconstruction.
• It suggests a complete unfolding of the
  alpha-helical protein to a beta-sheet form.
• However, the inter-chain disulfides are not
  disrupted.
• There are 2 beta-strands per insulin molecule.

Jimenez et al., Proc. Of Natl. Acad of Sci., 2002
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Beta-Helical Model
Kishimoto A et al., Biochem Biophys Res Commun.
2004
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Zipper-Spine Model
C-term
N-term
Ivanova et al., Proc Natl. Acad of Sci., 2005
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Domain Swapping

• The term Domain-Swapping was used for the first
  time in the context of the Diphtheria Toxin
  dimer.
• Domain also refers to a secondary structure
  element like an ?-helix or a ?-strand.

Bennett, M.J., Choe, S. and Eisenberg, D. (1994)
Prot. Sci. 3, 1444-1463
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Domain Swapping Models
With a Zipper Spine
Without a Zipper Spine
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Bovine Pancreatic Ribonuclease (RNase A)

• RNase A has four disulfides bridging Cys26-Cys84,
  Cys40-Cys95, Cys58-
• Cys110 and Cys65-Cys72. This severely restricts
  conformational change.
• RNase A forms two types of 3D domain-swaps when
  lyophilized from 40 acetic
• acid.
• One of the two catalytic His residues of RNase
  A(His 12) is on the core domain
• whereas the other (at position 119) is on the
  swapped domain. This segregation
• of catalytic His on different domains offers the
  possibility of forming an active
• complementary dimer from two inactive monomers.

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Ribonuclease A
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Minimum Polar Zipper at the Open Interface
Liu Y., et al., Nat. Struct. Biol., 2001
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Monomer, Dimer and Model for the Fiber
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Testing the Model
Introduction of the glutamine residues in the
hinge-loop regions of RNase A
Confirm by EM that the Gln-expanded RNase A
forms amyloid fibers. Test for Congo Red
birefringence and Cross-Beta diffraction.
Examine if fibers contain domain-swapped function
al RNase A molecules that have a native-like
fold.
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Activity Assays
A119
H119
A12
H12
H
H
A119
H119
A12
H12
A119
A12
H119
H12
A
A119
A
H119
A12
H12
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Principle Governing the Activity Assay
Quencher
Flourophore
DNARNA bases
Excite at 490nm
Quencher
Fluoresces
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Other Constructs
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Structural Model for RNase A fibrils
Antiparallel sheets formed by PolyQ tract
Functional, Domain swapped molecules
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Domain Swapping Zipper Spine Model
Spine
Steric Zipper
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Summary

• Structures of cross-? spines and the analysis of
  the RNase A fibrils support the idea that only
  limited regions of proteins are necessary for
  forming the amyloid core.
• The structures reiterate the importance of
  hydrogen bonding in the direction of fibril
  growth.
• The structures support the importance of
  sidechain interaction as demonstrated by the high
  shape complementarity in the dry interface.
• Domain Swapping Zipper Spine Model is one
  possible mechanism for fibril formation in RNase
  A and potentially other amyloid systems.

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Acknowledgments
Shiho Tanaka Zeynep Ashalin Oztug
Jennifer Warfel
Stuart Sievers
Martin Phillips
Alex Lisker
Duilio Cascio