Hsp90 as a regulator of protein conformation and function

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
Structure of Hsp90
15-2

• 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, 239-250.
3
15-3
Structure of HtpG dimerization domain
Harris et al. (2004) Structure 12, 1087-1097.
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15-4
Structure of HtpG
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 chaperonesubstrate 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
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15-5
Stirling et al. (2006) Nat. Struct. Mol. Biol.
6
Physiological targets of Hsp90
15-6

• 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), hy
poxia-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
15-7

• 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 modulate
s 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
15-8

• 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
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Hsp90-HOP-Hsp70 interaction
15-9

• 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, 199-210.
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TPR motif specificity of HOP
15-10
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?
15-11

• 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