Protein folding in the cell: The Hsp90 Chaperone...
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1 Protein folding in the cell: The Hsp90 Chaperone Machine Stefan Rüdiger Utrecht, 26 November2009 Master Molecular and Cellular Life Sciences 2009/2010 Master course “Molecular recognition” R116 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
Transcript of Protein folding in the cell: The Hsp90 Chaperone...
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Protein folding in the cell: The Hsp90 Chaperone Machine
Stefan Rüdiger
Utrecht, 26 November2009
Master Molecular and Cellular Life Sciences 2009/2010
Master course “Molecular recognition”
R116!
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
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Unscrambling egg white with Guanidinium Chloride
Hermsen et al (2006)!
Buffer 7 M GdmCl 7 M GdmCl heat
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
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Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp
Specialised chaperones
Many heat shock proteins are chaperones
Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp
Specialised chaperones
Many heat shock proteins are chaperones
Hsp100 Hsp90 Hsp70 Hsp60 Hsp40 sHsp
Specialised chaperones
4 humans
1 E. coli
Many heat shock proteins are chaperones
Hsp90α Hsp90β
HtpG
cytosol
ER
mitochondria Trap1
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
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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 receptors Transcription factors
p53 CFTR
5. Hsp90 - unknowns
1. Ratio mismatch
Hsp90 ?
~20000 genes ~20000 proteins
~130 Hsp90 substrates ~60 kinases
Steroid receptors Transcription factors
p53 CFTR
2. Substrate diversity
Hsp90 ?
~20000 genes ~20000 proteins
~130 Hsp90 substrates ~60 kinases
Steroid receptors Transcription factors
p53 CFTR
3. What is up with kinases?
Hsp90 ?
~20000 genes ~20000 proteins
~130 Hsp90 substrates ~60 kinases
Steroid receptors Transcription factors
p53 CFTR
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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 & Gewirth!Mol Cell 28:41-56 (2007)0!
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SAXS indicates Hsp90 flexibility
Krukenberg, Förster, Rice, Sali & Agard!Structure 16:755-765 (2008)!
7. Hsp90 interaction with substrates
CRINEPT-TROSY
Hsp90 can be monitored by NMR
158 µM!Hsp90!Dimer!(15N,2H)!
110!
130!
125!
120!
115!
15N!
10! 9! 8! 7!1H! 10! 9! 8! 7!1H!
110!
130!
125!
120!
115!
15N!
HSQC spectrum of free p53 core domain
p53core (free) HSQC
100 µM!p53core!(15N,2H)!
100 µM!p53core!(15N,2H)!
10! 9! 8! 7!1H!
110!
130!
125!
120!
115!
15N!
p53core bound to Hsp90 is unfolded!
147 µM!Hsp90!Dimer!(2H)!
p53core (Hsp90) CRINEPT-TROSY!
100 µM!p53core!(15N,2H)!
100 µM!p53core!(15N,2H)!
10! 9! 8! 7!1H!
110!
130!
125!
120!
115!
15N!
147 µM!Hsp90!Dimer!(2H)!
p53core (urea) HSQC!p53core (Hsp90) CRINEPT-TROSY!
100 µM!p53core!(15N,2H)!
Signals of urea-unfolded p53 cluster in same region
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p53core bound to Hsp90
…is unfolded
Ile labelling allows NMR approach
Full length
Elif Karagöz Afonso Duarte
Hans Ippel
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
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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-Targeted Library - Chemdiv · HtpG (high-temperature protein G), whereas Archaebacteria lack a Hsp90 representative [24]. All eukaryotes possess cytosolic members, called Hsp90](https://static.fdocuments.in/doc/165x107/5c687a8609d3f2f5638b9b2b/hsp90-targeted-library-htpg-high-temperature-protein-g-whereas-archaebacteria.jpg)
