Protein Folding in the Cell 3

BIOC 212 Winter 2013 Jason C. Young

HSP70 Cycle

HSP70-ATP Substrate Binding: HSP70 DNAJ Co-chaperone interaction with HSP70 no yes DNAJ

HSP70-ADP yes no (transfer to HSP70) NEF

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
GroES

GroEL

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 the same ring (top) and opposite ring (bottom)
Apical domain down Apical domain up

Apical domain

ATPase domain

opposite ring interface

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 polar surface substrate can be encapsulated in cavity and folded
GroES apical domain cavity up position ATP/ADP cavity with polar (blue) surface polypeptide enclosed

apical domain

down position no nucleotide hydrophobic (yellow) exposed can bind polypeptide

GroEL Cycle
binds substrate with hydrophobic domains (no nucleotide top ring) ATP binding (top ring) encloses substrate inside a polar cavity under GroES 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) Nature Struc Mol Biol 16, 574-581

HSP70 and Chaperonins

human Hsc70 can act at early stages when substrate is extended; binds many substrates human chaperonin TRiC is specialized for folding certain proteins TRiC can act after Hsc70, at late stages of folding no direct contact between Hsc70 and TRiC

Young et al. (2004) Nature Reviews Mol. Cell Biol. 5, 781-791.

HSP90 Family
HSP90 chaperones are homodimers, with 2 identical subunits joined at 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

ATP dimerization

middle

C
co-chaperone p23 (pink)

Ali et al. (2006) Nature 440, 1013-1017

ATP

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

client proteins bound

E. coli Hsp90 (HtpG)

Shiau et al. (2006) Cell 127, 329340

HSP90 Substrate Binding

Yeast Hsp90

M+C open position

M+C closed position

M+C with substrate?

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.

HSP90 Proteins
compartment bacterial cytoplasm human cytosol human mitochondria human ER lumen HSP90 HtpG Hsp90, Hsp90 Hsp75 Grp94 co-chaperones no yes no no

* human Hsp90 and are both constitutively expressed and also induced by stress conditions

Multichaperone System
Human cytosolic Hsc70 and Hsp90 form a multichaperone system Many co-chaperones regulate Hsc70 and Hsp90 direct contact to chaperones many co-chaperones have modular domains provide flexibility folding and non-folding functions

Young et al. (2004) Nature Reviews Mol. Cell Biol. 5, 781-791.

Hsp90 Co-chaperones

ATPase Regulators

Kinase-specific Co-chaperone

TPR Domain Co-chaperones

Humans: Hsp90

p23, Aha1

Cdc37

Hop FKBP52 (PPIase) Tom70 many others

Hsp90 co-chaperones are mostly not essential but increase efficiency of function some also interact with Hsc70: Hop binds Hsc70 and Hsp90 at the same time

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

D, E
Hsc70 and Hsp90 are unrelated but have similar C-terminal sequence motifs: Hsc70: PTIEEVD-COO Hsp90: MEEVD-COOTPR domains recognize EEVD motifs Hsp70 C-terminal ionic, hydrogen bonds fragment

Hop TPR1 domain

TPR Co-chaperones
Hop has 2 TPR domains which bind the Hsc70 and Hsp90 motifs in a 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
Hsc70 Hsp90

Hop

TPR
Hsc70 or Hsp90

TPR
Hsp90 only
PPIase

ubiquitin ligase

CHIP

TPR

U-box

FKBP52

PPI

PPI

TPR

TPR Domain Co-chaperones

TPR domains are adaptors that connect chaperones to different protein complexes or locations FKBP52: PPIase, steroid receptor chaperone UNC-45: myosin-specific chaperone Tom70: mitochondrial import CHIP: ubiquitin ligase

Young et al. (2004) Nature Reviews Mol. Cell Biol. 5, 781-791.

Glucocorticoid Receptor
GR belongs to a family of steroid hormone receptors responds to hormones such as cortisol by activating and repressing specific genes anti-inflammatory function ligand binding domain recognizes hormone DNA binding domain binds to GR promoter elements (GRE) N-terminal activation domain regulates transcription

human GR 110 kDa

NTD

DBD

LBD ligand binding domain cortisol

N-terminal DNA binding activation domain domain

GR LBD
hydrophobic steroid is bound in the interior of the LBD, and is necessary 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 and activated

cortisol

Sequential Action of Chaperones

LBD is folded by a sequence of chaperones DnaJA1 (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

DnaJA1: human type 1 Hsp40

The Heat Shock Response

heat shock followed by recovery period triggers a typical response in human cells translation is inhibited immediately (minutes) and slowly recovers (~1 hour after HS) transcription of HSPs is up-regulated (6-12 hours); transcription of other proteins is down-regulated high expression of chaperones increased degradation of unfolded proteins

heat shock 45C

~1 h translation recovers

~12 h HSP expression highest

~24 h return to normal

37C recovery

HSF1
HSF1 transcription factor is regulator of the heat shock response DNA binding domain, trimerization domain, and transcription activation domain Inactive HSF1 is monomeric; active HSF1 is a trimer Active HSF1 recognizes HSE (heat shock element) promoters

Regulation of HSF
monomeric HSF1 is native, but mimics unfolded protein and is bound by the chaperone Hsp90 after heat shock, Hsp90 binds truly unfolded proteins, and HSF1 becomes 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
heat shock Active Hsf

Inactive HSF Hsp90

unfolded proteins

Voellmy (2004) Cell Stress Chaperones 9, 122-133.

Compare and Contrast

Substrate binding: HSP70 Chaperonin (top ring) HSP90 ATP ADP no nucleotide how does it bind substrate

GR vs. HSF1 chaperone binding, activation, inactivation

End of 3
Hartl et al. (2011) Molecular chaperones in protein folding and proteostasis. 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.