1. Hsp90 as a regulator of protein conformation and function
15-1
Hsp90 is an abundant eukaryotic protein
makes up about ~2% of cytosolic protein content
not surprising: number of proteins it interacts with is huge
~90 kDa in size; forms dimers
phosphoprotein
Hsp90 is also present in the ER, where it is termed Grp94, or Glucose-regulated protein 94kDa (most abundant protein of the ER)
production increased by cellular stresses, i.e. it is a heat-shock protein
it is essential for viability in both cytosol and endoplasmic reticulum, where it is ubiquitously present
Hsp90 exists in some bacteria but not archaea

2. target protein binding
15-2
Structure of Hsp90
domain structure of Hsp90:
N-terminal domain
middle domain
C-terminal domain N C
GA, ATP, target
protein binding
target protein
binding
dimerization site;
target protein binding
geldanamycin (GA) binds in ATP-binding pocket and prevents the activity of Hsp90; mutations in ATP binding site also prevent activity and leads to cell death in vivo
ATP-binding domain
(partial crystal structure)
Stebbins et al. (1997) Cell 89,

3. Structure of HtpG dimerization domain
15-3
Structure of HtpG dimerization domain
Harris et al. (2004) Structure 12,

4. Structure of HtpG Structure of Full-length Hsp90 (yeast Hsp90) 15-4
A Simplified Schematic of Possible Hsp90 Function
Substrate is represented as a green circle to mark the expected position of contacts, but not as a direct indication of substrate size or structural details. The bulk of the substrate may, in fact, be variously extended outside of the Hsp90 clamp with chaperone:substrate contacts limited to a subdomain or smaller structural element of the client protein. Also, binding of ATP is known to stimulate the association of the amino-terminal domains, but as the timing of hydrolysis and release is not yet well understood, these details have been excluded from these figures for simplicity. Substrate is presumed to bind within the Hsp90 dimer clamp, contacting multiple mobile hydrophobic elements including helix 2 of the carboxy-terminal domains (shown as cylinders) and loops or patches along the inner surface of the middle domain (not explicitly shown). The association of the amino-terminal domains, stimulated by ATP binding, occludes the inner volume and juxtaposes the hydrophobic features. We show three possible routes of substrate release (blue arrows). Reversal of the initial binding event may return the chaperone to its open state with amino terminal domains separated (left). Alternately, full closure of the clamp may be incompatible with substrate binding as the hydrophobic features are mutually masked (upper right). Finally, inspired by the GHKL family member topoisomerases, transient dissociation of the carboxy-terminal domains could cause the exposed hydrophobic dimer interface to compete for binding with helix 2, thereby synchronizing substrate release with an opening of the Hsp90 topological ring to permit substrate to exit the Hsp90 clamp (lower right).
Structure of Full-length Hsp90 (yeast Hsp90)
from Pearl and Promodou, Ann. Rev. Biochem. 2006

5. 15-5
Stirling et al. (2006) Nat. Struct. Mol. Biol.

6. Physiological targets of Hsp90
15-6
Physiological targets of Hsp90
Hsp90 interacts with, regulates the conformation of, and the activity of, a large variety of cell signalling molecules, transcription factors, cytoskeleton, etc.
Substrates
heat-shock factor (HSF) Hsp90 downregulates activity in conjunction with Hsp70 system
other transcription factors receptors (steroid, glucocorticoid),
hypoxia-inducible factor-1 (HIF-1), etc.
kinases tyrosine kinases (v-src, lck, insulin receptor, etc.)
serine-thronine kinases (eIF-2 kinase, v-raf, c-raf, etc.)
protein kinase CK-II
cytoskeleton actin, tubulin (protection during heat stress)

7. Hsp90-associated proteins
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Hsp90-associated proteins
it is believed that Hsp90 never functions in isolation in eukaryotes; it always appears to be associated with a variety of cofactors
Cofactor Notes
Hsp70 Hsp90 activity dependent on Hsp70 system (incl. Hsp40)
HOP HOP, Heat-shock Organizing Protein, brings Hsp70 and Hsp90 together via TPR interaction domains
p23 modulates ATPase activity of Hsp90
HIP co-chaperone of Hsp70
PPIases cyclophilin-40, FKBP51, FKBP52
others kinase-targeting co-chaperone Cdc37/p50

8. 15-8
Hsp90 functional cycle
Hsp90 not only assists the folding of proteins, but can also modulate the conformation/function of proteins
binding of steroid to Hsp90-bound steroid receptor releases the receptor in a form that can bind DNA and activate transcription
Hsp90
p23
FKBP
HIP
HIP
HIP
Hsp40
Hsp70
Hsp70
target
protein
(non-native/
non-functional)
Hsp70
HOP
HOP
FKBP
p23
Hsp90
Hsp90 is also involved in quality control: binding of denatured protein can lead either to its folding or its degradation
target
protein
(native/functional)
FKBP
p23

9. Hsp90-HOP-Hsp70 interaction
15-9
Hsp90-HOP-Hsp70 interaction
HOP contains multiple TPR motifs (tetratricopeptide) which can interact with a EEVD motif at the very C-terminus of proteins
TPR motifs are 34 amino acid degenerate sequences that occur in tandem, usually 3 copies or more in a row
these motifs are found in other proteins--
not simply those associated with molecular
chaperones
N-terminus
of MEEVD
each TPR has a
helix-turn-helix
structure
C-terminus
of MEEVD
molecular
surface of TPR
and peptide
- EEVD is a consensus motif (can vary somewhat)
- 3 TPR motifs needed for proper binding
Scheufler et al. (2000) Cell 101,

10. TPR motif specificity of HOP
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TPR motif specificity of HOP
Domain structure of HOP
Example ITC data; incubate one
component with another at different
ratios, measure heat
ITC data shows Hsp70/Hsp90 preference for TPR1 or TPR2A binding sites
n (ratio of binding) is ~1 for all
Kd measured (lower μM means tighter binding)

11. Is Hsp90 a ‘typical’ chaperone?
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Is Hsp90 a ‘typical’ chaperone?
Hsp90 can prevent the aggregation of a denatured protein upon dilution from a chaotrope (urea, guanidine hydrochloride)
but it is not very efficient compared to other chaperones
cannot refold a protein by itself
how can it recognize so many different proteins that belong to rather limited classes (e.g. all sorts of different transcription factors, kinases, etc.)
Susan Lindquist laboratory: Hsp90 as a capacitor for evolution