Protein Folding in the Cell – 3
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Transcript of Protein Folding in the Cell – 3
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Protein Folding in the Cell 3
BIOC 212
Winter 2013
J ason C. Young
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HSP70 Cycle
HSP70-ATP HSP70-ADP
Substrate Binding:
HSP70no yes
DNAJ yesno
(transfer to HSP70)
Co-chaperoneinteraction with HSP70
DNAJ NEF
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Chaperonins (HSP60 family)
Chaperonins are large protein complexes with multiple subunits
Typical double-ring structure
E. coli GroEL: 2 rings x 7 subunits x 60 kDa = 840 kDa
In some cases with cap co-chaperone
E. coli GroES cap: 7 subunits x 10 kDa
Act by enclosing substrates within cavity
GroEL
GroES
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GroEL Subunits
Each GroEL subunit has an ATPase domain and an Apical domain Base of the ATPase domain is interface with opposite ring
Movement of the Apical domain is controlled by nucleotide in thesame ring (top) and opposite ring (bottom)
Apical domain down Apical domain up
Apical domain
ATPasedomain
opposite ring
interface
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GroEL Apical Domains
Down position (no nucleotide): hydrophobic surface on apical domains is exposed
provide multiple binding sites for substrate
Up position (ATP and ADP):
hydrophobic surface now binds GroES cap cavity formed with polarsurface
substrate can be encapsulated in cavity and folded
down position no nucleotidehydrophobic (yellow) exposed
can bind polypeptide
up position ATP/ADP
cavity with polar (blue) surfacepolypeptide enclosed
apical domain
GroES
apical domain
cavity
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GroEL Cycle
binds substrate with hydrophobic domains (no nucleotide top ring)
ATP binding (top ring) encloses substrate inside a polar cavity underGroES cap substrate is not bound but is free inside cavity
confinement promotes folding
Substrate is enclosed while ATP is hydrolyzed to ADP no nucleotide (top ring) and ATP (bottom ring) release GroES and
substrate
multiple cycles
Hartl & Hayer-Hartl (2009) NatureStruc Mol Biol 16, 574-581
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HSP70 and Chaperonins
human Hsc70 can act at earlystages when substrate isextended; binds many substrates
human chaperonin TRiC is
specialized for folding certainproteins
TRiC can act after Hsc70, at latestages of folding
no direct contact between Hsc70and TRiC
Young et al. (2004) Nature ReviewsMol. Cell Biol. 5, 781-791.
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HSP90 Family
HSP90 chaperones are homodimers, with 2 identical subunits joinedat the C-termini
human Hsp90: 2 x 90 kDa = 180 kDa
dimer can open and close, like nutcracker
ATP controls opening and closing of the dimer
Ali et al. (2006) Nature 440, 1013-1017
ATP
dimerization
ATP
co-chaperone
p23 (pink)
N middle C
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HSP90 Conformation
Substrate can be bound in nucleotide-free and ATP-bound state
ATP binding allows dimer to close
ATP hydrolysis to ADP compacts the dimer and releases substrate
Shiau et al. (2006) Cell 127, 329340
E. coli Hsp90 (HtpG)
client proteinsbound
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M+Copen position
M+Cclosed position
M+Cwith substrate?
HSP90 Substrate Binding
Thought to bind near-native polypeptides at late stages of folding May bind to hydrophobic surfaces and support a flexible substrate
like a Scaffold
Substrate binding site may be between or alongside subunits
Ali et al. (2006) Nature 440, 1013-1017.
Yeast Hsp90
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HSP90 Proteins
compartment HSP90 co-chaperones
bacterial cytoplasm HtpG no
human cytosolHsp90,
Hsp90yes
human mitochondria Hsp75 no
human ER lumen Grp94 no
* human Hsp90 and are both constitutively expressed and also induced by
stress conditions
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Multichaperone System
Young et al. (2004) Nature ReviewsMol. Cell Biol. 5, 781-791.
Human cytosolic Hsc70 andHsp90 form a multichaperonesystem
Many co-chaperones regulateHsc70 and Hsp90
direct contact to chaperones
many co-chaperones havemodular domains
provide flexibility
folding and non-foldingfunctions
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Hsp90 Co-chaperones
Hsp90 co-chaperones are mostly not essential but increaseefficiency of function
some also interact with Hsc70:
Hop binds Hsc70 and Hsp90 at the same time
ATPaseRegulators
Kinase-specificCo-chaperone
TPR Domain
Co-chaperones
Humans:
Hsp90 p23, Aha1 Cdc37
Hop
FKBP52 (PPIase)
Tom70
many others
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Human Hsp90 ATPase Cycle
Substrate is transferred from Hsc70 to Hsp90 (nucleotide-free)
Hop binds both Hsc70 and Hsp90 to organize transfer Hsp90 in the ATP state binds substrate tightly
co-chaperone p23 stabilizes the closed ATP state of Hsp90
ATP hydrolysis releases substrate from Hsp90
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D, E
Hsc70 and Hsp90 are unrelated but have similar C-terminalsequence motifs:
Hsc70: PTIEEVD-COO- Hsp90: MEEVD-COO-
TPR domains recognize EEVD motifs ionic, hydrogen bonds Hsp70 C-terminal
fragment
Hop TPR1 domain
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TPR Co-chaperones
Hop has 2 TPR domains which bind the Hsc70 and Hsp90 motifs ina similar way, but are still specific for each
modular TPR domains are found in other co-chaperones
recognize Hsc70 or Hsp90 or both
not dependent on nucleotide state of Hsc70 or Hsp90
Hop TPR TPR
Hsc70 Hsp90
CHIP TPR U-box
Hsc70 or Hsp90
ubiquitin ligase
FKBP52 PPI TPR
PPIase
Hsp90 only
PPI
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TPR Domain Co-chaperones
TPR domains are adaptors thatconnect chaperones to differentprotein complexes or locations
FKBP52: PPIase, steroidreceptor chaperone
UNC-45: myosin-specificchaperone
Tom70: mitochondrial import
CHIP: ubiquitin ligase
Young et al. (2004) Nature ReviewsMol. Cell Biol. 5, 781-791.
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Glucocorticoid Receptor
GR belongs to a family of steroid hormone receptors
responds to hormones such as cortisol by activating and repressingspecific genes anti-inflammatory function
ligand binding domain recognizes hormone
DNA binding domain binds to GR promoter elements (GRE) N-terminal activation domain regulates transcription
cortisol
NTD DBD LBD
N-terminal
activation domain
DNA binding
domain
ligand binding
domain
human GR110 kDa
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GR LBD
hydrophobic steroid is bound in the interior of the LBD, and isnecessary to maintain the native structure
in the absence of hormone, LBD cannot fold stably
chaperones keep the LBD partially folded, able to bind hormone
other domains of GR are normally fully folded
partially folded,unstable, inactive
native andactivated
cortisol
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Sequential Action of Chaperones
LBD is folded by a sequence of chaperones
DnaJ A1 (Hsp40) / Hsc70
Hsc70 / Hop / Hsp90
Hsp90 / FKBP52 / p23
without hormone, GR continues to cycle through chaperone system with hormone, GR becomes a stable dimer and active
DnaJ A1: human type 1 Hsp40
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The Heat Shock Response
heat shock followed by recovery period triggers a typical response inhuman cells
translation is inhibited immediately (minutes) and slowly recovers(~1 hour after HS)
transcription of HSPs is up-regulated (6-12 hours); transcription ofother proteins is down-regulated
high expression of chaperones
increased degradation of unfolded proteins
37C
45C
heat shock
recovery
~1 htranslationrecovers
~12 hHSP expression
highest
~24 hreturn tonormal
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HSF1
HSF1 transcription factor is regulator of the heat shock response
DNA binding domain, trimerization domain, and transcriptionactivation domain
Inactive HSF1 is monomeric; active HSF1 is a trimer
Active HSF1 recognizes HSE (heat shock element) promoters
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Regulation of HSF
Voellmy (2004) Cell Stress Chaperones 9, 122-133.
monomeric HSF1 is native, but mimics unfolded protein and isbound by the chaperone Hsp90
after heat shock, Hsp90 binds truly unfolded proteins, and HSF1becomes free to trimerize and activate transcription
chaperones including Hsp90 are expressed and help refold or
degrade misfolded proteins HSF1 is down-regulated by binding of excess Hsp90 to the
monomer form
Hsp90
Inactive HSF
unfoldedproteins
Active Hsfheat
shock
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Compare and Contrast
GR vs. HSF1
chaperone binding, activation, inactivation
Substratebinding:
ATP ADP no nucleotidehow does it
bind substrate
HSP70
Chaperonin
(top ring)
HSP90
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End of 3
Hartl et al. (2011) Molecular chaperones in protein folding andproteostasis. Nature 475, 324-332.
Young et al. (2004) Pathways of chaperone mediated protein folding
in the cytosol. Nature Reviews Mol. Cell Biol. 5, 781-791.