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
2
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.
3
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
4
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.