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Protein folding in the cell: The Hsp90 Chaperone Machine
Stefan Rüdiger
Utrecht, 26 February 2009
Master Biomolecular Sciences 2008/2009Master course “Membrane Biogenesis, Protein
Folding and Sorting”
R099
1. Protein folding and unfolding
Protein unfolding
Un-boiling egg white with Guanidinium Chloride
Unscrambling egg white with Guanidinium Chloride
Unscrambling egg white with Guanidinium Chloride
Unscrambling egg white with Guanidinium Chloride
Hermsen et al (2006)
Buffer 7 M GdmCl 7 M GdmClheat
2. Protein folding in the cell
The Central Dogma (Crick 1958)
DNA
RNA
Protein
Biochemical Pathways
3. Molecular Chaperones
Processes involving chaperones
degradation
translocation
folding aggregation
Hsp100Hsp90Hsp70Hsp60Hsp40sHsp
Specialised chaperones
Many heat shock proteins are chaperones
Hsp100Hsp90Hsp70Hsp60Hsp40sHsp
Specialised chaperones
Many heat shock proteins are chaperones
Hsp100Hsp90Hsp70Hsp60Hsp40sHsp
Specialised chaperones
4 humans
1 E. coli
Many heat shock proteins are chaperones
Hsp90Hsp90
HtpG
cytosol
ER
mitochondriaTrap1
Grp94
4. Hsp90 - knowns
Hsp90
N N
M M
C C• 83 KDa• 166 KDa dimer• ATPase in N-terminal domain• Substrate binding site unknown
The Hsp90 substrate pool
Hsp90?
~20000 genes ~20000 proteins
The Hsp90 substrate pool
Hsp90?
~20000 genes ~20000 proteins
~130 Hsp90 substrates
The Hsp90 substrate pool
Hsp90?
~20000 genes ~20000 proteins
~130 Hsp90 substrates
~60 kinases
Steroid receptorsTranscription factors
p53
CFTR
5. Hsp90 - unknowns
1. Ratio mismatch
Hsp90?
~20000 genes ~20000 proteins
~130 Hsp90 substrates
~60 kinases
Steroid receptorsTranscription factors
p53
CFTR
2. Substrate diversity
Hsp90?
~20000 genes ~20000 proteins
~130 Hsp90 substrates
~60 kinases
Steroid receptorsTranscription factors
p53
CFTR
3. What is up with kinases?
Hsp90?
~20000 genes ~20000 proteins
~130 Hsp90 substrates
~60 kinases
Steroid receptorsTranscription factors
p53
CFTR
6. Hsp90 structures
Hsp90-N (human)
Stebbins, Russo, Schneider, Rosen, Hartl & Pavletich, Cell 89:239-250 (1997)
Hsp82 (yeast Hsp90; AMP-PNP, p23)
Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:1013-1017
HtpG (E. coli Hsp90, ATP-free)
Shiau, Harris, Southworth & Agard, Cell 127:329-340 (2006)
The Hsp90 substrate cycle
Richter & Buchner Cell 127:251-253 (2006)
Hsp90 conformations
Dollins, Warren, Immormino & GewirthMol Cell 28:41-56 (2007)0
SAXS indicates Hsp90 flexibility
Krukenberg, Förster, Rice, Sali & AgardStructure 16:755-765 (2008)
7. Hsp90 interaction with substrates
CRINEPT-TROSY
Hsp90 can be monitored by NMR
158 µMHsp90Dimer(15N,2H)
110
130
125
120
115
15N
10 9 8 71H
10 9 8 71H
110
130
125
120
115
15N
HSQC spectrum of free p53 core domain
p53core (free) HSQC
100 µMp53core(15N,2H)
100 µMp53core(15N,2H)
10 9 8 71H
110
130
125
120
115
15N
p53core bound to Hsp90 is unfolded
147 µMHsp90Dimer(2H)
p53core (Hsp90) CRINEPT-TROSY
100 µMp53core(15N,2H)
100 µMp53core(15N,2H)
10 9 8 71H
110
130
125
120
115
15N
147 µMHsp90Dimer(2H)
p53core (urea) HSQC
p53core (Hsp90) CRINEPT-TROSY
100 µMp53core(15N,2H)
Signals of urea-unfolded p53 cluster in same region
p53core bound to Hsp90
…is unfolded
4 humans
1 E. coli
Kinase targeting: Hsp90, cdc37 (targeting factor) and Cdk4 (substrate)
Vaughan, Gohlke, Sobott, Good, Ali, Prodromou, Robinson, Saibil & Pearl Mol Cell 23:697-707 (2006)
8. The Hsp90 cycle
The Hsp90 substrate cycle
Richter & Buchner Cell 127:251-253 (2006)
9. Hsp90 co-factors
Substrate targeting factor Cdc37
Roe, Ali, Meyer, Vaughan, Panaretou, Piper, Prodromou & Pearl Cell 127:329-340 (2006)
4 humans
1 E. coli
Kinase targeting: Hsp90, cdc37 (targeting factor) and Cdk4 (substrate)
Vaughan, Gohlke, Sobott, Good, Ali, Prodromou, Robinson, Saibil & Pearl Mol Cell 23:697-707 (2006)
ATP hydrolysis: Hsp90 and Aha1
Meyer, Prodromou, Liao, Roe, Vaughan, Vlasic, Panaretou, Piper, & Pearl EMBO J 23:1402-1410 (2004)
4 humans
1 E. coli
Nucleotide exchange: Hsp90 and p23
Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:1013-1017
Coupling to degradation: Hsp90 and CHIP
Zhang, Windheim, Roe, Peggie, Cohen, Prodromou & Pearl Mol Cell 20:525-538 ()2005)
The Hsp90 substrate cycle
Richter & Buchner Cell 127:251-253 (2006)
Hsp90-dependend refolding of citrate synthase
Jakob, Lilie, Meyer, Buchner JBC 270:7288-7294 (1995)
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