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

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Hsp90 as a regulator of protein Hsp90 as a regulator of protein conformation and functionconformation and function

domain structure of Hsp90:

ATP-binding domain(partial crystal structure)

Stebbins et al. (1997) Cell 89, 239-250.

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

N CN-terminal domain middle domain C-terminal domain

GA, ATP, targetprotein binding

target proteinbinding

dimerization site;target protein binding

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Structure of Hsp90Structure of Hsp90

Structure of HtpG dimerization domainStructure of HtpG dimerization domain15-3

Harris et al. (2004) Structure 12, 1087-1097.

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Structure of Full-length Hsp90 (yeast Hsp90)

A Simplified Schematic of Possible Hsp90 FunctionSubstrate 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 HtpGStructure of HtpG

from Pearl and Promodou, Ann. Rev. Biochem. 2006

Stirling et al. (2006) Nat. Struct. Mol. Biol.

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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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Physiological targets of Hsp90Physiological targets of Hsp90

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-associated proteinsHsp90-associated proteins

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

targetprotein(non-native/non-functional)

Hsp40

Hsp70

HIP HIP

HOP

Hsp70

HIP

HOP

Hsp70

Hsp90p23FKBP

Hsp90p23

FKBP

p23 FKBPtargetprotein(native/functional)

Hsp90 is also involved in quality control: binding of denatured protein can lead either to its folding or its degradation

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Hsp90 functional cycleHsp90 functional cycle

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 molecularchaperones

- EEVD is a consensus motif (can vary somewhat)- 3 TPR motifs needed for proper binding

N-terminusof MEEVD

C-terminusof MEEVD

each TPR has ahelix-turn-helixstructure

molecularsurface of TPRand peptide

Scheufler et al. (2000) Cell 101, 199-210.

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Hsp90-HOP-Hsp70 interactionHsp90-HOP-Hsp70 interaction

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)

Domain structure of HOP

Example ITC data; incubate onecomponent with another at differentratios, measure heat

15-10TPR motif specificity of HOPTPR motif specificity of HOP

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

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Is Hsp90 a Is Hsp90 a ‘‘typicaltypical’’ chaperone? chaperone?