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Hsp90 as a regulator of protein
conformation and function
 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 Glucoseregulated 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
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Structure of Hsp90
 domain structure of Hsp90:
N-terminal domain middle domain
N
GA, ATP, target
protein binding
target protein
binding
C-terminal domain
C
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, 239-250.
Structure of HtpG dimerization domain
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Harris et al. (2004) Structure 12, 1087-1097.
Structure of HtpG
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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
Structure of Full-length Hsp90 (yeast Hsp90) carboxy-terminal domains could cause the exposed hydrophobic dimer
interface to compete for binding with helix 2, thereby synchronizing substrate
from Pearl and Promodou, Ann. Rev. Biochem. 2006
release with an opening of the Hsp90 topological ring to permit substrate to
exit the Hsp90 clamp (lower right).
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Stirling et al. (2006) Nat. Struct. Mol. Biol.
Physiological targets of Hsp90
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 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)
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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
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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
Hsp40
target
protein
(non-native/
non-functional)
HIP
Hsp70
 Hsp90 is also involved in
quality control: binding of
denatured protein can lead either
to its folding or its degradation
HIP
Hsp90
p23
FKBP
HIP
Hsp70
Hsp70
HOP
HOP
p23
target
protein
(native/functional)
p23
FKBP
Hsp90
FKBP
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
N-terminus
tandem, usually 3 copies or more in a row
of MEEVD
 these motifs are found in other proteins-not simply those associated with molecular
chaperones
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, 199-210.
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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)
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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