Protein folding in the cell: The Hsp90 Chaperone Machine Stefan Rüdiger Utrecht, 26 February 2009 Master Biomolecular Sciences 2008/2009 Master 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

Hermsen et al (2006) Buffer7 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

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 Hsp90  Hsp90  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

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

6. Hsp90 structures

Hsp90-N (human) Stebbins, Russo, Schneider, Rosen, Hartl & Pavletich, Cell 89: (1997)

Hsp82 (yeast Hsp90; AMP-PNP, p23) Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:

HtpG (E. coli Hsp90, ATP-free) Shiau, Harris, Southworth & Agard, Cell 127: (2006)

The Hsp90 substrate cycle Richter & Buchner Cell 127: (2006)

Hsp90 conformations Dollins, Warren, Immormino & Gewirth Mol Cell 28:41-56 (2007)0

SAXS indicates Hsp90 flexibility Krukenberg, Förster, Rice, Sali & Agard Structure 16: (2008)

7. Hsp90 interaction with substrates

CRINEPT-TROSY Hsp90 can be monitored by NMR 158 µM Hsp90 Dimer ( 15 N, 2 H) N H

H N HSQC spectrum of free p53 core domain p53core (free) HSQC 100 µM p53core ( 15 N, 2 H)

100 µM p53core ( 15 N, 2 H) H N p53core bound to Hsp90 is unfolded 147 µM Hsp90 Dimer ( 2 H) p53core (Hsp90) CRINEPT-TROSY 100 µM p53core ( 15 N, 2 H)

100 µM p53core ( 15 N, 2 H) H N 147 µM Hsp90 Dimer ( 2 H) p53core (urea) HSQC p53core (Hsp90) CRINEPT-TROSY 100 µM p53core ( 15 N, 2 H) 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: (2006)

8. The Hsp90 cycle

The Hsp90 substrate cycle Richter & Buchner Cell 127: (2006)

9. Hsp90 co-factors

Substrate targeting factor Cdc37 Roe, Ali, Meyer, Vaughan, Panaretou, Piper, Prodromou & Pearl Cell 127: (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: (2006)

ATP hydrolysis: Hsp90 and Aha1 Meyer, Prodromou, Liao, Roe, Vaughan, Vlasic, Panaretou, Piper, & Pearl EMBO J 23: (2004)

4 humans 1 E. coli Nucleotide exchange: Hsp90 and p23 Ali, Roe, Vaughan, Meyer, Panaretou, Piper, Prodromou & Pearll Nature 440:

Coupling to degradation: Hsp90 and CHIP Zhang, Windheim, Roe, Peggie, Cohen, Prodromou & Pearl Mol Cell 20: ()2005)

The Hsp90 substrate cycle Richter & Buchner Cell 127: (2006)

Hsp90-dependend refolding of citrate synthase Jakob, Lilie, Meyer, Buchner JBC 270: (1995)