Molecular Chaperones

BIOC 450

23 November, 2009

Jason C. Young

History

• 1973: spontaneous folding; sequence and structure

• 1989: chaperone hypothesis

• 1991: GroEL pure system

• 1993: DnaK (Hsp70) pure system; J domain family

• 1997: Hsp90 ATPase; GroEL mechanism

• 1998-2000: TPR domains

• 2001: human genome project

• 2001-present: co-chaperone “explosion”

• Native structure is compact and stabilized by multiple hydrophobic contacts

• In a folding reaction, the native state has the lowest free energy– protein folding is spontaneous in principle– in practice, impractically slow

• Folding intermediates are flexible, less compact, with exposed hydrophobicity

Native State

cytochrome B562 with Haem

Model of folding reactionDaggett and Fersht

Folding vs. Aggregation

• Folding intermediates can aggregate with other unfolded polypeptides

• Both folding and aggregation depend on hydrophobic interactions

• Normal aggregates are structurally disordered

• Amyloid fibrils are a special type of aggregate– ordered conformation that

is not the native state– pathogenic

Dobson (2003) Nature 426, 884-890.

Folding Thermodynamics

• each point on curve represents a different polypeptide conformation• each conformation has a different G• a curve is only one of many possible folding paths

G

number of internal contactscompactnesscoverage of hydrophobicity

native

intermediate

unfolded

Dobson (2003) Nature 426, 884-890

Folding Landscape

• many different unfolded and partially folded states

• different folding pathways lead to one native state

• intermediates can persist in local free energy minima

– “kinetically trapped”, requires energy to escape minimum

Folding Landscape

Native State:Global Minimum

Local Minimum

Folding / Aggregation Landscape

• multiple intermolecular contacts in aggregates can make them more stable than individual native state

• amyloid aggregates are the most stable

How Do Chaperones Help Folding?

• In theory, chaperones could:– prevent aggregation– prevent off-pathway intermediates– unfold off-pathway intermediates to

re-start folding– increase on-pathway rates

X

+X

X

X

+ +

off-pathway intermediate(kinetically trapped)

on-pathway intermediate leads to native state

apical domain

ATPase domain

Bukau & Horwich (1998) Cell 92, 351-366

Chaperonins

down: no GroES bound, hydrophobicity exposed

up: GroES bound, polar surface exposed

GroES cofactor

Apical domain

ATPase domain

ATPase domain

Apical domain

GroELE. coli GroEL

14 identical subunits

2 rings of 7 subunits

GroEL Folding Cycle

• binding to apical domains• stretching – partial unfolding• confinement

– prevents aggregation– favours compact over extended conformations

Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581

• “Polypeptide Clips” – monomers which bind short, extended hydrophobic regions of substrate

• ATPase domain allosterically controls substrate binding domain • substrate binding domain opens and closes

DnaK (E. coli)

extended conformation

Hsp70 Proteins

ATPase domain(NBD)

Substrate binding domain (SBD)

Hsp70 Proteins

• human Hsc70 is constitutively expressed, Hsp70 is inducible– also have forms in human mitochondria, ER lumen

• co-chaperones: J domain proteins, and nucleotide exchange factors (NEF)

compartment Hsp70J domain co-

chaperoneNEF

E. coli cytoplasm DnaK DnaJ GrpE

human cytosolHsc70

Hsp70 (inducible)

Hsp40

DJA1, DJA2

others

various

Hsp70 States

• biochemical, biophysical, structural data• ATP-bound:

– NBD and SBD locked together – SBD pulled open

• ADP-bound:– NBD and SBD separate– SBD shut, substrate binding

• two-state mechanism conserved inall Hsp70 proteins

• J domains bind Hsp70s and are necessary to stimulate ATP hydrolysis

• Type 1 DnaJ proteins have J domains and substrate binding domains

• Some proteins have J domains but do not bind substrate – activate Hsc70 for specialized functions

J Domain Co-chaperones

substrate binding dimerizationJ domain

Other J domain co-chaperones

Type 1 co-chaperonesyeast Ydj1 (Type 1)

Ramos et al. J Mol Biol (2008) 383, 155-166

Hsp70 Cycle

• DnaJ binds substrate• J domain stimulates ATP hydrolysis by Hsp70

– substrate binding by Hsp70 in ADP state• NEFs cause ATP re-binding and release of substrate

Hartl & Hayer-Hartl (2009) Nature Struc Mol Biol 16, 574-581

Human Hsc70

• pure protein system: Hsc70, unfolded luciferase• DnaJA2 essential • NEF Hsp110 not essential, but enhances• model to study mechanism of Hsc70 and DnaJ

How Does Hsc70 Help Folding?

• In theory, chaperones could:– prevent aggregation– prevent off-pathway intermediates– unfold off-pathway intermediates to

re-start folding– increase on-pathway rates

• which of these apply?

X

+X

X

X

+ +

Aggregation Prevention

• Hsc70 binding to hydrophobic intermediate prevents interactions with other unfolded polypeptide

• evidence: expect an optimal rate of binding and release• NEFs can be adjusted to an optimal ratio

Confinement

• GroEL encloses substrate entirely in cavity– prevents larger intermediates from forming– mutations on inside change folding activity

• monomeric Hsc70 not large enough• Hsc70 and DnaJ could bind at once

* *

*mutations inside cavity

GroEL

Unfolding / Annealing

• GroEL: substrate binding domains move apart before enclosing• fluorescence spectroscopy

– substrate becomes less compact during GroEL binding, then more compact after enclosure

– partial unfolding– not always productive

• Hsc70 and DnaJ could bind at once

Hsc70 and DnaJ Coordination

• human DnaJA2 and A1 are very similar• J domains and substrate binding domains are functional• DJA2 supports folding in model, DJA1 does not; chimera inactive• domain coordination must be different

– DJA1 function in cells – different folding challenge?

DnaJ Transfer to Hsc70

• binding of substrate by both DnaJ and Hsc70 during transfer• double binding could change substrate conformation

– compress substrate (confinement) – pull apart (partial unfolding)

Increase Folding Rate

• analogy to enzyme: reduction of free energy barrier on pathway

• barrier could be rotational freedom around peptide bond

• Hsc70 binding could promote rotation• evidence: synthetic peptide

isomerization• role of DnaJ + +

The Hsc70-Hsp90 Multichaperone System

Hsc70 and Hsp90:ATP-dependent “folding machines”

TPR domains: adaptors that recognize Hsc70 or Hsp90

FKBP52: peptidyl-prolyl isomerase

CHIP: E3 ubiquitin ligase

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

(NEF)

The Chaperone-Tom70 Pathway

Hsp90

Hsc70

Tom70

preprotein

Tom40 Import Pore

Tim9/10

ATP GANB

Bag

Young et al. (2003) Cell 112, 41-50Fan et al. (2006) J. Biol. Chem. 281, 33313-33324.

novobiocin, geldanamycin: Hsp90 inhibitorsBag: Hsc70 NEF

Chaperone-Tom70 Mechanisms

• precursors are inner membrane proteins• Hsc70 could:

– prevent aggregation– prevent off-pathway intermediates– unfold off-pathway intermediates to

re-start folding– increase on-pathway rates – no– coordinate with Tom70 for transport

across membrane?

off-pathway intermediate(kinetically trapped)

X

Tom70Tom40

Questions

Hsp90Hsc70 DnaJ