David Agard Laboratory UCSF -...

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Trapping Dynamic Conformational States and CochaperoneInteractions of the Hsp90 Molecular Chaperone

Daniel Southworth

David Agard LaboratoryUCSF

Apoptosisp53IP6K2ASK1

NOSeNOSnNOS

kinasessrcaktraf

PolymerasesTelomeraseViral PolymerasesRev. Transcriptase

centrosomePolo kinase

Mitochondrial Import

Client Proteins>150

Steroid Hormone ReceptorsProgesteroneEstrogenGlucocorticoid

General Protein Folding

Hsp90: A molecular chaperone that activatesspecific substrate client proteins

Hop

Hsp70

p23

Immunophilins

Aha1

Cochaperones>20

p50 (Cdc37)

Sse1

Hsp90

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

Apo ExtendedE. coli Hsp90

Shiau, et al 2006

p23

Closed, ATP conformationYeast Hsp90:AMPPNP:p23-Ali MM, et al. 2006

Compact ADP DimerModeled from E. coli Hsp90:ADP

tetramerShiau, et al 2006

NTDMD

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. coliHsp90

Three Nucleotide-Stabilized ConformationalStates in E. coli Hsp90

150 Å

Negative stain w/Uranyl FormateDefocus: 1.5 nM120 keV20 Å resolution

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YeastHsp90

Apo ATP ADP

Hsp90 nucleotide-dependent conformationsappear to vary between species

E. coliHsp90

HumanHsp90

Are the mechanisms fundamentally different?

Apo human Hsp90 + AMPPNP

Using glutaraldehyde to crosslink and traptransiently sampled states

Glutaraldehyde (0.005%)

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

Crosslinked States are Nucleotide Dependentand Match EM Structures

150 Å

E.coliADP

modelSingle

ParticlesRF

Avgs.

Human Hsp90ADP

SingleParticles

RFAvgs.

Yeast Hsp90ADP

E.coliADP

modelHsp90:ATP

modelSingle

ParticlesRF

Avgs.

Human Hsp90AMPPNP

Hsp90 Chaperone Cycle Involves ThreeUniversally Conserved Conformational States

•Hsp90 chaperone cycle involves a unique conformational equilibrium thatis different between species.

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Frac

tion

of H

sp90

E. coli ATP

Yeastapo

YeastATP

E. coliapo

Quantitation of the open and closed stateconformational equilibrium

Human apo

Human ATP

Match single particle data to aset of unique 2D projections ofthe open and closed structuresof Hsp90

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

The Human Hsp90 Chaperone Cycle Involves CoordinatedInteractions with Specific Co-Chaperones

Client Loading

ATP

Client Maturation

Complexes Dissociate: Low affinity and transient

Hsp90 alone180 kDa

Hsp90:Hop250 kDa

Mol

ar M

ass

(Dal

tons

)

Volume (ml)

Norm

alized Refractive Index

Hsp90 alone180 kDa

CrosslinkedHsp90:Hop300 kDa

CysCys

Stable 300 kDa Hsp90:Hop tetramer complexformed following disulfide crosslinking

Hop60 kDa

Cys+

light scattering/chromatography

100 Å

0.005% Glutaraldehyde

Hsp90:Hop

HOPHOP

HOP HOP

Negative-stain EM of 300 kDa Hsp90:Hop

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4k x 4k Image1.19 Å/pixel

30 nM

30 nM

8k x 8k Image1.19 Å/pixel

150 Å

5.0 µM defocus

Cryo-EM of Hsp90:Hop

Technai TF20120 KevTietz 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 Åresolution

FSC

ATP N-MiddleConformation

Apo Middle-CConformation +

Hsp90:Hop:Hsp70

Disulfide Crosslinking Stabilizes Multiple Hsp90 Complexes

360 kDa Client

DVEE

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 improveshomogeneity 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 AgardYifan ChengShawn ZhengJustin Kollman

AMI, Scripps San DiegoBridget CarragherClint PotterJoel Quispe

FundingAmerican Cancer SocietyHHMI