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


1
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

2
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
3
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
4
Hallmarks of Amyloid
Long, Unbranched Fibrils
4.77Å
11.7Å
Bind Congo Red and yield an apple green
birefringence
5
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?

6
Structures of the Cross-? Spines
Balbirnie at al., Proc Natl Acad Of Sci., 2001
7
GNNQQNY forms Amyloid-like fibrils and Related
Microcrystals
8
Christian Riekel
Anders Madsen
9
Data collection at ESRF
10
Microcrystal diffraction at ESRF
Scale Bar 10µm
11
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12
GNNQQNY Structure
Nelson et al., Nature 2005
13
Hydrogen Bonding
14
Interfaces Between Sheets
High Shape Complementarity
15
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.

16
Important Hexapeptide Motifs in Tau
Von Bergen et al., 2005
17
Rosetta Energy Predictions for Tau
Thompson et al., Proc. Natl Acad of Sci., 2006
18
Fibrils
Von Bergen et al., 2001
19
Structure of VQIVYK
Val1
Tyr5
Ile3
Val4
Lys6
Gln2
20
Tyr5
Ile3
Val1
Val4
Lys6
Gln2
21
Hydrogen Bonding
22
Interfaces
Dry Interface
Wet Interface
23
Hetero-Interfaces
Q
K
I
Y
V
I
Q
I
V
V
K
Q
24
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
25
Beta-Helical Model
Kishimoto A et al., Biochem Biophys Res Commun.
2004
26
Zipper-Spine Model
C-term
N-term
Ivanova et al., Proc Natl. Acad of Sci., 2005
27
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
28
Domain Swapping Models
With a Zipper Spine
Without a Zipper Spine
29
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.

30
Ribonuclease A
31
Minimum Polar Zipper at the Open Interface
Liu Y., et al., Nat. Struct. Biol., 2001
32
Monomer, Dimer and Model for the Fiber
33
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.
34
Activity Assays
A119
H119
A12
H12
H
H
A119
H119
A12
H12
A119
A12
H119
H12
A
A119
A
H119
A12
H12
35

Principle Governing the Activity Assay
Quencher
Flourophore
DNARNA bases
Excite at 490nm
Quencher
Fluoresces
36
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37
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38
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39
Other Constructs
40
Structural Model for RNase A fibrils
Antiparallel sheets formed by PolyQ tract
Functional, Domain swapped molecules
41
Domain Swapping Zipper Spine Model
Spine
Steric Zipper
42
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.

43
Acknowledgments
Shiho Tanaka Zeynep Ashalin Oztug
Jennifer Warfel
Stuart Sievers
Martin Phillips
Alex Lisker
Duilio Cascio
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