Scenarios for Protein Aggregation Illustrations using A peptides and PrP C as examples Ruxandra I....
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![Page 1: Scenarios for Protein Aggregation Illustrations using A peptides and PrP C as examples Ruxandra I. Dima F. Massi (Columbia) D. Klimov (GMU) J. Straub.](https://reader038.fdocuments.us/reader038/viewer/2022110323/56649d635503460f94a46106/html5/thumbnails/1.jpg)
Scenarios for Protein Aggregation
Illustrations using A peptides and PrPC as examples
Ruxandra I. Dima F. Massi (Columbia)D. Klimov (GMU)J. Straub (BU)B. Tarus (BU)M. S. Li (Poland)
(PrPC)
A-peptides
DIMACS meeting Rutgers University April 20, 2006
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Energy landscape for monomeric folding
Monomer can misfold to multiple conformations
Structural variations in the CBAs are imprinted in oligomers and fibrils
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Aggregation Linked to diseases Protein deposition diseases
* transmissible spongiform encephalopathies (TSE; Mad Cow Disease) * Alzheimer’s disease, Parkinson’s disease * diabetes (type II)
All these diseases = related to misfolding and protein aggregation
Misfolding into multiple amyloid conformations (strains)
Examples: prion proteins (TSE), Alzheimer’s, CWD
Question: What is the nature of the initial events in oligomer formation?
Two broad scenarios: Illustrations using A peptides and PrPC
Current AD hypothesis: Soluble oligomers are neurotoxic
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Scenarios for Fibrillization
N* = metastable
N* formation = partial unfolding
A and TTR
Prions
N* = stable
N* formation in prions = unfolding of N
(D.T., D. Klimov and R.Dima, Curr. Opin. Struct. Biol., 2003)
KG dependson rate of formation ofN* from N orU
PrPc is metastablewith respect to PrP*
aggregation proneparticle
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Cascade of events to Fibrils
Scenario I (Partial unfolding/ordering)
nA16-22 (A16-22)nPolydisperseOligomers
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Heterogeneous Nucleation and Growth
On + kM
Differing
Supra-molecular
Assembly
Heterogeneous Nuclei
KG = F(Seq,C,GC)
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A-peptide in vivo is a metabolic product of precursor protein
• Alzheimer’s Disease (AD) is responsible for 50% of cases of senile dementia
• A-peptide is a normal byproduct of metabolism of Amyloid Precursor Protein (APP)
• Cleavage of APP results from action of specific proteases called secretases
• In Selkoe’s “A hypothesis,” AD is a result of the accumulation of A-peptide
many naturally occurring mutants E22Q “Dutch” mutant
A10-35
A1-40 and A1-42 peptides
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A16-22 For Scenario I
• Mechanism and Assembly Pathways• Sequence Effects• Role of water• Fragment has CHC • Interplay of hydrophobic/electrostatic effects
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Trimer Structurefrom MD
Antiparallel sheets
Monomer is a Random Coil
Structure: Inter-peptide Interaction Driven
Interior is dry:Desolvation an early event
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Dominant assembly pathway involves-helical intermediate
Teplow JMB 2001
“Effective confinement”induces helix formation
-helical intermediate“entropically” stabilized
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Origin of -helical Intermediate
Case I
C C* C* = Overlap concentration
Low Peptide Concentration
Rjk
Rjk ≈ C-(1/3)
Rjk/Rg 1
Polypeptide is mostly a random coil
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C C* Peptides Interact
j
k
Rjk / Rg ~ O(1)
Peptide j is entropicallyconfined
In peptide j confinement induces transient structure
For A16-22 interaction drives transient -helix formation
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Hydrophobic andcharged residuesstabilize oligomers
-OOC
+NH3
NH3+
COO-+NH3
COO-
1
2
3
Anti-parallel registry satisfiesHydrophobic and charged interactions
Principle of Organization
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Structural orientation requires charged residues
K16G/E22G trimer is unstable
Kinetics and stability of Oligomerization determined By balance of hydrophobic andCharged interactions
Enhanced growth kinetics in E22Qdue to change in charged statesMassi,Klimov,DT, Straub (2002)
“Long-range” correlations between charged residues in protein families linked to disease-related proteins (Dima and DT, Bioinformatics (2003)
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Electrostatics interactions essential in amyloid formation: Charged states
A10-35-NH2E22QA10-35-NH2
• E22Q “Dutch” mutant peptide shows enhanced rate of amyloid formation@
• Lower propensity for amyloid formation in WT peptide due to Glu- charged states (versus Glno)• Proposed INVERSE correlation between charge and aggregation rate - now seen experimentally%
*Zhang et al. Fold. Des. 3:413 (1998).@ Miravalle et. Al., J. Biol. Chem., 275, 27110-27116 (2000).#Massi and Straub, Biophys. J. 81:697 (2001); Massi, Klimov, Thirumalai and Straub, Prot. Sci. 11:1639 (2002).% Chiti, Stefani, Taddei, Ramponi and Dobson, Nature 424:805 (2003).
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Templated assembly
Seed = Trimer
Insert A16-22 monomer
Tetramer forms rapidly
Nucleus 4
Barrier to addition
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Important structural motifs in Apeptide monomer and fibrils
• A-peptide structure determined in aqueous solution by NMR by Lee and coworkers*
• Monomer A10-35 peptide has well-defined “collapsed coil” structure
• Collapsed coil is stabilized by VGSN turn region and LVFFA central hydrophobic cluster#
* S. Zhang et al., J.Struct. Biol. 130, 130-141 (2000). # Massi, Peng, Lee and Straub, Biophys. J. 80:31 (2001).% Tycko and coworkers, PNAS 99: 16742 (2002).
VGSN turn region
central hydrophobic LVFFA cluster
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
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Scenario II (Global unfolding of PrPC)
N* = metastable
N* formation = partial unfolding
A, TTR Prions
N = metastable
N* formation involves global unfolding of N
KNN* depends on sequence and G† between N and N*
(D.T., D. Klimov and R.D., Curr. Op. Struct. Biol., 2003)
PrPSc growth kineticsDepends on rate ofNN* transition KNN*
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Mechanism of assembly and propagation
Prions normal form PrPC = mostly -helical
scrapie form PrPSc = mostly strand• the “protein-only hypothesis”: (Prusiner et al., Cell 1995 and Science 2004)
PrPSc = template to catalyze conversion of normal form into the aggregate
βαFluctuation β*
NucleationGrowth
ββ
Propagation by recruitment
?PrPC*
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Question and Hypothesis
23190 121
Minimal infectious unit
Disordered in PrPC
Ordered
PrPSc
(48% β, 25% α)
(45% α, 8% β)
Unfolded
PrPC*
?
PrPC
Proposal:
PrPSc formation is preceded by transition from
α PrPC* state
(20% α)
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NMR Structure of Cellular form (PrPC)
• PrPC: 45% , 8%
• PrPSc(90-231): 25% 48%
mPrPC(121-231)
(Cys179-Cys214)
• Prions: “…Prion is a proteinaceous particle that lacks nucleic acid”
(Prusiner, PNAS, 1998)
(Caughey et al. Biochemistry 30, 7672 (1991))
Wuthrich 1997
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H1 in mammalian PrPC is helical
Charge patterns in H1 is rarely found in PDB, E. Coli and
Yeast genomes
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Pattern search for H1 in PrPC
(i,i+4) = oppositely charged residues
search sequences of 2103 PDB helices (Lhelix ≥ 6)
(i,i+4) salt-bridges in mPrPC
Random considerations:
10),(
),(
n
LR helix
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Sequence analysis shows PrPC H1 is a helix
- X - - + X X + - X
search PDBselect (1210 proteins)
23 (1.9%) sequences
83% = α-helical, 17% = random coil
search E. Coli (4289 proteins) genome
51 (1.2%) sequences
search yeast (8992 proteins) genome
253 (2.8%) sequences
Pattern of charged residues in H1 is unusual and NEVER associated with β-strand
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Experiments and MD simulations show H1 is very stable
Conformational fluctuations and stability of H1 with two force
fields
Stability is largely due to the three salt bridges in the 10 residue H1 from mPrPC
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High helical propensity at all positions in H1 H1 from mPrP (10 residues)
positions 144-153
• 773 TIP3P water, 30 ǺT cubic box, 300 K, neutral pH
• 5 trajectories, 85 ns
MOIL package (Amber and OPLS) (R. Elber et al.)
Helix
Strand
PDB
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Unusual hydrophobicity pattern in H2
X X X H H X X H H X H X H X X X X H P P P P X
search PDBAstral40 (6000 proteins)
12 (0.2%) sequences
the sequence is NEVER entirely α-helical
(last 5 residues = non-helical in 87% of cases)
search E. Coli (4289 proteins) genome
46 (1%) sequences
search yeast (8992 proteins) genome
122 (1.4%) sequences
Pattern of hydrophobicity of H2 is rare and NEVER entirely in a α-helix
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H2+H3 in mammalian PrPC frustrated in helical state
Conformational fluctuations in H2+H3 implicate a role for second
half of H2 in the PrPC
PrPC* transition
R. I. Dima and DT Biophys J. (2002); PNAS (2004)
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Structural transitions in H2+H3
NAMD package (Charmm)
H2+H3 in mPrP , S-S bond
H2 starts to unwind around position 187
unwinding by stretching and bending
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X-ray structure of PrPC dimer shows
changes in H2 and H3 Domain-swapped dimer of
huPrPC (Surewicz et al., NSB 8, 770, 2001)
H1: 144-153
(monomer: 144-153)
H2: 172-188 and 194-197
(monomer: 173-194)
H3: 200-224
(monomer: 200-228) PDB file 1i4m
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Rarely populated PrPC* shows changes in H2 and H3
15N-1H 2D NMR under variable pressure and NMR relaxation analysis on shPrP(90-231)
(James et al., Biochemistry 41, 12277 (2002) and 43, 4439 (2004))
in PrPC* C-terminal half of H2 and part of H3 are disordered
98.99% 1.0% 0.01%
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Many pathogenic mutations are clustered around H2 and H3
H2 and H3 regionFrom Collinge (2001)
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Scenario for initiation of PrPC aggregation
PrPSc
(48% β, 25% α)
(45% α, 8% β)
Unfolded
PrPC*
PrPC
(20% α)
Finding:
transition α PrPC* state
initiated in second half of H2 and does not involve H1
G†
G† /KBT 1
PrPC* formation improbable
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Proposed structures for PrPC*
Charmm
H1 still α-helical
H3 only partially α-helical
Amber and OPLS
PDB
(48% α-helix)
(30% α-helix)
(20% α-helix)
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Conclusions
• Multiple routes and scenarios for fibril formation• Electrostatic and hydrophobic interactions determine structure and
kinetics• Conformational heterogeneity in N* controls oligomer and fibril
morphology (may be relevant for strains)• Phase diagram (T, C) plane for a single amyloidogenic protein is
complex due to structural variations in the misfolded N*
• Templated growth occurs by addition of one monomer at a time• Nucleus size and growth mechanism depends on protein