1. Protein Folding and Molecular Chaperones

2. Protein Folding and Diseases
Contents
Protein Folding
Molecular Chaperones
Protein Folding and Diseases

3. 1. Protein Folding

4. Protein-Folding Problem
1958
John Kendrew et al., published the first structure of a globular protein, myoglobin.
“ Perhaps the most remarkable features of the molecule are its complexity and its lack of symmetry”
1962
Nobel prize in Chemistry was awarded to Max Perutz and John Kendrew.
Now
~80,000 structures in protein database (PDB)

5. Common Structural Patterns
Motifs, folds, or supersecondary structures
Stable arrangements of several elements of secondary structure
Domains
Stable, globular units

6. Classification of Protein Structures
Structural classification of proteins (SCOP) database
Classification
All a
All b
a/b : a and b segments are interspersed or alternate
a + b : a and b regions are segregated
~1,200 different folds or motifs
Protein family (~4,000)
Proteins with similarities in
Primary sequence
(and/or) Structure
Function
Superfamily
Families with little primary sequence similarity but with similarities in motifs and function

7. Structural classification from SCOP database

8. Structural classification from SCOP database

9. Amino Acid Sequenc Determines Tertiary Structure
Amino acid sequence contains all the information required to protein folding
First experimental evidence by Christian Anfinsen (1950s)
Denaturation of ribonuclease with urea and reducing agent
Spontaneous refolding to an active form upon removal of the denaturing reagents

10. Protein Folding is not a trial-and-error process
E. coli
make 100 a.a. protein in 5 sec
10 possible conformations/ a.a.
 conformations
10-13 sec for each conformation
 1077 years to test all the conformations

11. Protein folding problem has not yet been solved
The physical folding code
How is the 3D structure determined by the physicochemical properties encoded in the amino acid sequence?
The folding mechanism
How can proteins fold so fast even with so many possible conformations?
Predicting protein structure using computers
Can we devise a computer algorithm to predict 3D structure from the amino acid sequnece?

12. The Physical Code of Protein Folding
Weak Interactions
Hydrogen bond
Hydrophobic interactions
Van der Waals interactions
Electrostatic interactions
Backbone angle preferences
Chain entropy
Large loss of chain entropy upon folding
Covalent bonding
Disulfide bonding

13. The Rate Mechanisms of Protein Folding
Models for protein folding
Hierarchical folding
From local folding (a helix, b sheets) to entire protein folding
Molten globule state model
Initiation of folding by spontaneous collapse by hydrophobic interactions

14. Thermodynamics of Protein Folding
Free-energy funnel
Unfolded states
High entropy and high free energy
Folding process
Decrease in the number of conformational species (entropy) and free energy
Semistable folding intermediates

15. Computing Protein Structures
Computer-based protein-structure prediction competition
Critical Assessment of protein Structure Prediction (CASP) in every second summer since 1994

16. Computing Protein Structures
Template-modeling (homology modeling, comparative modeling)
Structure prediction based on the structure of a protein with a sequence homology.
Free modeling (ab initio, de novo modeling)
Fragment assembly
PDB search of overlapping fragments of target proteins
Assembly of fragments using some scoring functions
Successful for short proteins (<100 a.a.)

17. Computing Protein Structures

18. 2. Molecular Chaperones

19. Nuclear import/export
Molecular Chaperones
Proteins facilitating protein folding, transport, and degradation
AAAA
proteasome
Folding
Translocation
Degradation
Nuclear import/export
Misfolding

20. Classes of Molecular Chaperones
Ribosome-associated chaperones
Trigger factor (prokaryotes)
RAC, NAC (eukaryotes)
Cytosolic chaperones
Hsp70
Induced in stressed cells (heat shock protein)
Binding to hydrophobic regions of unfolded proteins, preventing aggregation
Cyclic binding and release of proteins by ATP hydrolysis and cooperation with co-chaperones (Hsp40 etc.)
E. coli: DnaK (Hsp70), DnaJ (Hsp40)
Hsp90
Small Hsps
Chaperonin
Protein complex providing microenvironments for protein folding
E. coli : 10~15% protein require GroES (lid) and GroEL
Eukaryotes: TriC/CCT
Organelle-specific chaperones (eukaryotes only)
ER chaperones
Mitochondrial chaperones

21. Isomerases in Protein Folding
Protein disulfide isomerase (PDI)
Shuffling disulfide bonds
Peptide prolyl cis-trans isomerase (PPI)
Interconversion of the cis and trans isomers of Pro peptide bonds

22. Co-translational Folding
Prokaryotes
Trigger factor
Cyclic association and dissociation with ribosome
Binds to hydrophobic regions of newly synthesized polypeptide chains
Shields nascent chains from degradation by proteases
Improve the yields of correctly folded model substrates by reducing the speed of folding
Eukaryotes
Hsp70 and J-protein–based systems
Ribosome-associated complex (RAC)
Heterodimeric nascent polypeptide-associated complex (NAC).

23. Trigger Factor

24. Molecular Chaperones

25. Protein Folding by DnaK and DnaJ

26. Chaperonin in Protein Folding

27. Roles of Hsp70 and Hsp90 Misfolding Folding Assembly Hsp70 Hsp90
AAAA
Misfolding
Hsp70
Folding
Hsp90
Metastable client proteins
Assembly

28. Hsp90 HtpG Hsp82, Hsc82 Hsp90a, Hsp90b (cytosol) Grp94 (ER)
Prokaryotes
HtpG
Yeast
Hsp82, Hsc82
Higher eukaryotes
Hsp90a, Hsp90b (cytosol)
Grp94 (ER)
Trap-1 (mitochondria) M N C
Acidic linker
HtpG, Trap-1
Other Hsp90s

29. Hsp90 Chaperone Network in Yeast
Genes interacting with Hsp90
~200 physical interactions
~451 genetic and chemical-genetic interactions
Zhao, R. et al., 2005, Cell
McClellan A.J. et al., 2007, Cell

30. Hsp90 Chaperone Cycle Open ATP-bound ATP ADP + Pi ATP hydrolysis
Lid C M N
ATP hydrolysis
Lid open : ADP bound
Figure 1 Working model of the conformational cycle of Hsp90. In the initial step (1), the open form of Hsp90 (protomers shown in light blue and medium blue) is charged with a client protein (yellow), aided by the Hsp70-Hsp40 machinery and the adaptor protein HOP, yielding an intermediary complex3. At this stage, the client protein presumably approaches a near-native conformation, resulting in dissociation of Hsp70. Subsequently, HOP is probably displaced by other TPR-clamp cochaperones such as the immunophilins Cyp40/Cpr6, FKBP51 and FKBP52 (ImPh, green). Binding of ATP to Hsp90 (2) triggers conformational changes in the N domain, ultimately resulting in N-domain dimerization and formation of a closed state, with composite active sites being formed between N and M domains of the same protomer. This closed state of Hsp90 is further stabilized by p23/Sba1 (orange)13. At this stage, steroid-receptor client proteins become primed for hormone binding and thus full activation. The cycle is completed by hydrolysis of ATP (3), which may be triggered by Aha1 (pale orange), resulting in the release of client protein and restoration of the open state of Hsp90
Closed
Lid close : ATP bound

31. Class of Hsp90 Co-chaperones
Higher eukaryotes
Yeast
Function
With TPR
Hop
Sti1
Adaptor to Hsp70
Cyp40
Cpr6, Cpr7
Peptidyl-prolyl isomerase
FKBP51, FKBP52 -
Sgt1
Adaptor for SCF and client proteins
PP5
Ppt1
Phosphatase
Without TPR
Aha1
Aha1, Hch1
Activation of Hsp90 ATPase activity
p23
Sba1
Inhibition of Hsp90 ATPase activity
Cdc37
Adaptor for kinases
Chp-1, Melusin
Unknown

32. TPR Motif One TPR motif contains two antiparallel a-helices
Protein-protein interaction module
One TPR motif contains two antiparallel a-helices
Tandem array of TPR motifs generate a right-handed helical structure
TPR domain in co-chaperones binds to MEEVD sequence in the Hsp90 C-terminus
Hsp90 MEEVD
PP5 TPR

33. Regulation of ATPase Activity by Co-chaperones
ADP + Pi
Sgt1 (CS domain)
Hsp90 N (O)
ATP
Hsp90 N (O)
Cdc37 ( )
Hsp90 N (C)
Aha1 (1-153)
Sba1

34. Protein Folding and Aggregation

35. Conditions Inducing Protein Aggregation
Mutations prone to aggregate
Huntington’s disease
Familial forms of Parkinson’s disease and Alzheimer’s disease
Defects in protein biogenesis
Translational errors
Assembly defects of protein complexes
Environmental stress conditions
Heat shock
Oxidative stress
Aging

36. Deposition of Aggregates
Bacteria
Inclusion body
Yeast
Juxtanuclear quality-control compartment (JUNQ)
Soluble, misfolded, ubiquitylated proteins
Perivacuolar insoluble protein deposit (IPOD).
Insoluble, terminally aggregated
Mammals
Aggresome

37. Protein Disaggregation
Hsp70-Hsp104 (ClpB) bi-chaperone
Hsp70-J protein
Transfer aggregates to Hsp104
Hsp104
Threading activity to refold aggregate

38. 3. Protein Folding and Diseases

39. Protein-Folding Diseases
Amyloidoses
Diseases caused by formation of insoluble amyloid fibers

40. Protein-Folding Diseases
Cystic fibrosis
Misfolding of cystic fibrosis transmembrane conductance regulator (CFTR)
Neurodegenerative diseases
Alzheimer’s, Parkinson’s, Huntinton’s disease, ALS
Prion diseases
Mad cow disease (bovine spongiform encephalopathy, BSE)
Kuru, Creutsfeldt-Jakob disease in human
Scrapie in sheep
Prion : proteinaceous infectious only protein
PrPSc (scrapie) prion form converts PrPC to PrPSc

41. Formation of Amyloid Fibers
Amyloid –b peptide

42. Chaperones as Drug Targets
Hsf1
Transcriptional activation of heat shock proteins
Activators of Hsf1 as drugs for protein-folding diseases
Hsp70
Hsp90
Clients proteins include some oncoproteins
Hsp90 inhibitors as cancer drugs

43. Hsp90 Client Proteins in Cancer
Roles of Hsp90 client proteins in cancer
Her2, Raf-1, Akt
Self-sufficiency in growth signals
Plk, Wee1, Myt1
Insensitivity to antigrowth signals
RIP, Akt
Evasion of apoptosis
hTERT
Limitless replicative potential
Hif-1a, Fak, Akt
Sustained angiogenesis
Met
Tissue invasion and metastasis

44. Hsp90 Inhibitors as Anti-Cancer Drug
Protein stabilization
Cancer
Hsp90 inhibitor
Oncoprotein
Her2,Raf-1,Akt, Hif-1a, survivin, mutant p53

45. Bended Form of ATP & 17-DMAG in the Pocket
- Hsp90 & ATP : bended formation Figure 2. Structure of the Hsp90•17-DMAG Complex
J.M. Jez et al, 2003, Chemistry & Biology

46. Hsp90 Inhibitors
Geldanamycin
Radicicol
17-AAG
ATP
PU3