1 Hsp90 nucleotide-dependent conformations Using glutaraldehyde to - - PDF document

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1 Hsp90 nucleotide-dependent conformations Using glutaraldehyde to - - PDF document

Nov 13, 2022 366 likes •437 views

Hsp90: A molecular chaperone that activates specific substrate client proteins Client Proteins >150 Trapping Dynamic Conformational States and Cochaperone General Protein Apoptosis Interactions of the Hsp90 Molecular Chaperone

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Trapping Dynamic Conformational States and Cochaperone Interactions of the Hsp90 Molecular Chaperone Daniel Southworth

David Agard Laboratory UCSF

Apoptosis p53 IP6K2 ASK1 NOS eNOS nNOS kinases src akt raf Polymerases Telomerase Viral Polymerases

  • Rev. Transcriptase

centrosome Polo kinase Mitochondrial Import

Client Proteins >150

Steroid Hormone Receptors Progesterone Estrogen Glucocorticoid General Protein Folding

Hsp90: A molecular chaperone that activates specific substrate client proteins

Hop Hsp70 p23 Immunophilins Aha1 Cochaperones >20 p50 (Cdc37) Sse1 Hsp90

Full-Length Crystal Structures of 180 kDa Hsp90 Dimer Identify Different Conformational States

Apo Extended
  • E. coli Hsp90
Shiau, et al 2006

p23

Closed, ATP conformation Yeast Hsp90:AMPPNP:p23
  • Ali MM, et al. 2006
Compact ADP Dimer Modeled from E. coli Hsp90:ADP tetramer Shiau, et al 2006

NTD MD CTD

  • Are these conformations conserved between species?
  • How are the conformational states coupled to client activation?
  • How does the human Hsp90 chaperone cycle function?

Apo ATP ADP

  • E. coli

Hsp90

Three Nucleotide-Stabilized Conformational States in E. coli Hsp90

150 Å

Negative stain w/ Uranyl Formate Defocus: 1.5 nM 120 keV 20 Å resolution
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Yeast Hsp90 Apo ATP ADP

Hsp90 nucleotide-dependent conformations appear to vary between species

  • E. coli

Hsp90 Human Hsp90

Are the mechanisms fundamentally different?

Apo human Hsp90 + AMPPNP

Using glutaraldehyde to crosslink and trap transiently sampled states

Glutaraldehyde (0.005%)

  • Glut. (0.005%) + protein (200 nM), 37° for 15 minutes. Stopped w/ 20 mM Tris.

Crosslinked States are Nucleotide Dependent and Match EM Structures

150 Å E.coli ADP model Single Particles RF Avgs. Human Hsp90 ADP Single Particles RF Avgs. Yeast Hsp90 ADP E.coli ADP model Hsp90:ATP model Single Particles RF Avgs. Human Hsp90 AMPPNP

Hsp90 Chaperone Cycle Involves Three Universally Conserved Conformational States

  • Hsp90 chaperone cycle involves a unique conformational equilibrium that

is different between species.

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Fraction of Hsp90
  • E. coli
ATP Yeast apo Yeast ATP
  • E. coli
apo

Quantitation of the open and closed state conformational equilibrium

Human apo Human ATP

Match single particle data to a set of unique 2D projections of the open and closed structures

  • f Hsp90
  • Conformational states are isoenergetic.
  • Nucleotide binding shifts inherent conformational equilibrium.

The Human Hsp90 Chaperone Cycle Involves Coordinated Interactions with Specific Co-Chaperones

Client Loading ATP Client Maturation

Complexes Dissociate: Low affinity and transient

Hsp90 alone 180 kDa Hsp90:Hop 250 kDa

Molar Mass (Daltons) Volume (ml) Normalized Refractive Index

Hsp90 alone 180 kDa Crosslinked Hsp90:Hop 300 kDa

Cys Cys

Stable 300 kDa Hsp90:Hop tetramer complex formed following disulfide crosslinking

Hop 60 kDa

Cys

+

light scattering/chromatography 100 Å

0.005% Glutaraldehyde Hsp90:Hop

HOP HOP HOP HOP

Negative-stain EM of 300 kDa Hsp90:Hop

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4k x 4k Image 1.19 Å/pixel

30 nM 30 nM

8k x 8k Image 1.19 Å/pixel

150 Å 5.0 µM defocus

Cryo-EM of Hsp90:Hop

Technai TF20 120 Kev Tietz 8k Camera

18.5 Å Cryo-EM Reconstruction of Hsp90:Hop

  • Data collected at Scripps (Leginon).
  • 15,000 particles (10,000 in final model).
  • Defocus: 1.5 to 3.5 µM.
  • CTF correction (CTFFIND).
  • Refinement using SPIDER, C2 symmetry.
0.054

1/Å

18.5 Å resolution

FSC

ATP N-Middle Conformation Apo Middle-C Conformation

+

Hsp90:Hop:Hsp70

Disulfide Crosslinking Stabilizes Multiple Hsp90 Complexes

360 kDa Client D V E E

Hsp70 FKBP52

TPR 300 kDa

Hsp90:AMPPNP:FKBP52 20 Å negative-stain reconstructions. Client Maturation ATP

Hsp90:Hop Hsp90:Hop:Hsp70 Hsp90:AMPPNP:FKBP52

Client Loading

Conclusions

  • Significant information can be gained from low-resolution structures
  • Glutaraldehyde crosslinking at low concentrations for short times improves

homogeneity and traps transient states.

  • Disulfide crosslinking in known binding sites improves stability.
  • Information about biological function gained from sorting heterogeneos data.
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Acknowledgements

David Agard Yifan Cheng Shawn Zheng Justin Kollman AMI, Scripps San Diego Bridget Carragher Clint Potter Joel Quispe

Funding

American Cancer Society HHMI