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