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)