Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 1

1

Prof. Pierre Goloubinoff

Towards a Unifying Mechanism

for the Hsp70 Chaperones

2

Nature Reviews Neuroscien

ce

6: 11

-22

(20

05)

Hsp70 chaperonescarry many apparently dissimilar, cellular functions

3

mRNA

Folding intermediat es

Energy

0

100

The life of a protein,

from birth to death, is full of events

Spontaneous

native folding

Anfinsen

Percentage of residue

of protein

in native conformation

Discrete folding

intermediates

Aggregates

Disease

Str

ess,

mu

tati

on

s

Molecular

chaperones

Proteases

Aggregates

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 2

4

UnfoldedMonomer

Native stateFolded monomeractive or inactive

Physiologicconditions

“Alter-native” stateDiscrete oligomer

Ribosome

ATP

The native and alter-native pathway

ATPATP Stressful & pathogenic conditions

Fibrillar, amyloid stateNon-discrete compact oligomerinactive, less-toxic, insoluble

Misfolded stateSoluble monomer

inactive, pathogenic

Aggregated stateNon-discrete loose

soluble oligomer

inactive, pathogenic

Stability

The misfolding and aggregating pathway

Hinault MP et al., J. Mol. Neurosci., 2006, 30(3): 249-65

Protease

5

Dictionary: A (social) chaperone is an older lady

that accompanies younger ladies to social events

to prevent “improper” associations

Ellis, 1988 (modified): A molecular chaperone is a protein

that accompanies nascent or stress-destabilized proteins

to prevent “improper” associations with other proteins,

such as protein aggregates

Goloubinoff, 2006: A molecular chaperone is a protein

that can proof-read protein structures and identify

abnormal misfolded or alternatively-folded protein surfaces;

Some can passively prevent misfolding and aggregation,

others use ATP to dissociate active oligomers

or scavenge toxic aggregates

What is a molecular chaperone?

Hinault and Goloubinoff, P., (2006), Adv. Exp. Med. Biol., 594: 47-54

6

Some, but not all, molecular chaperones

are heat-inducible proteins (HSPs)

42.5oC

Heat-shock

in the presence of 35S-met

Some, but not all, molecular chaperones

are heat-inducible proteins (HSPs)

Hwa Dai et al., Bot. Bull. Acad. Sin., (1996), 37(4): 261-264

28oC

Auto-radiogram

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 3

7

Hsp100Hsp90Hsp70Hsp60

Small HspsHsp60

GroEL

Small HSPs

IbpA/B

ATP ATP

ATP

The major

chaperone classes:

Hsp100

ClpB Hsp90

HtpG

ATP

8

The structure and function

of a very ancient chaperone: HSP70/DnaK

ATPLid

Base

9

Hsp70 can prevent misfolded proteins

to form larger aggregates

Stableaggregates

X

mRNA

NativeFolding intermediateMisfoldedmonomer

HSP70

Mogk et al., The EMBO Journal, (1999), 18: 6934–6949

Hsp70 (DnaK) knockout30°C 42°C

9%

16%pH 3.0 pH 10.0 pH 3.0 pH 10.0

9%

16%

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 4

10

Why does ATP-hydrolysis, v irtually “lock” Hsp70 on its substrate,

with a 1000 fold higher affinity

than in the “aggregation-prev enting” bound state???

The C-terminal protein-binding domainof Hsp70 (DnaK) can bind non-bulky hydrophobic residues

flanked by positiv e charges in extended peptides

Rudiger S, et al., EMBO J., 1997 Apr. 1, 16(7): 1501-7

11

Hsp70 needs

co-chaperones:

Hsp40 or DnaJ in E. coli

Triggers ATP hydrolysis, thus the locking of Hsp70

onto its substrate

Binds to misfolded proteins

Complex between the ATPase domain of DnaK and a GrpE homodimer (Harrison et al., Science, (1997), 276: 431-435)

A nucleotide

exchange factor:

GrpE in E. coli

J 1 2 3 4 C-terminus

N

4

13

2

HPD

II

IN

III C

G/FCys repeats

IVC

12

Aggregate

Spontaneousaggregation

“Client”

DnaJbinding

1

J

Unfolded segment

DnaK-binding

2ATP

ADP

DnaJ-acceleratedATP-hydrolysis,DnaK lockingDnaJ dissociation

3

4

GrpE-acceleratedDnaK unlocking

Spontaneousnative refolding

5

Native

The traditional HSP70 cycle: assisted-refolding by prevention of aggregation

6

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 5

13

Hsp70 alone (with DnaJ and GrpE)can solubilize and reactivate stable protein aggregates

Diamant et al., 2000

Glucose-6-P-dehydrogenase

G6

PD

H r

efo

ldin

g (

µM

)

Skowyra et al., 1990

RNA polymerase

BSAPe

rce

nt

RN

AP

ac

tiv

ity

Time at 30°C (min)Time (min)

14

Several Hsp70 (DnaK) molecules

cooperate in the reactivation of G6PDH aggregates

Yet, DnaK (Hsp70) acts as monomers !

Ben-Zvi et al., JBC, 2004, 279: 37298-303

15

The traditional HSP70 cycle:assisted-refolding by prevention of aggregation

This mechani sm does not explai n

how the energy of ATP hydrolysis may be used

by the chaperone to conver t stable protein

aggregates into unstable refolding intermediates

that can in turn convert into stable native species

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 6

16

3000 m

ATP

3003 m

1500 m

To be able to mediate the conversion

of stable protein aggregates

into even more stable native proteins,

ATPase chaperon es must be able

to actively unfold misfolded structures

in the aggregated proteins

17

The specific (refolding) activityof the chaperone increases with the substrate

dilution instead of decreasing

The specific (refolding) activityof the chaperone increases with the substrate

dilution instead of decreasing

Constant high DnaK,decreasing substrate

The chaperone/substrate ratio determines the chaperone activity

Ben-Zvi et al., JBC, 2004, 279: 37298-303

of substrate

Dilution

18

Hsp70 unfolds misfolded proteins

The specific unfolding activity of DnaK is optimal

at substoichiometric concentrations

Unfolding

Ben-Zvi et al., JBC, 2004, 279: 37298-303

Unfolding(Inactivation)

Refolding

(Reactivation)

Misfolding

Unfolding

Aggregation

Disaggregation

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 7

19Disaggregation depends on the aggregate size:

the larger the aggregates the sooner they are fragmented by DnaK

The Hsp70 disaggregating activity is sensitive to the size of the substrate

Rapid

Less rapid

Slow

Very slow

Ben-Zvi et al., JBC, 2004, 279: 37298-303

Unfolding(Inactivation)

Refolding

(Reactivation)

Misfolding

Unfolding

Aggregation

Disaggregation

20

Size doesmatter !

21

Aggregate size may affect

a Brownian unfolding machine

With large aggregates,

Brownian movement

of individual Hsp70 molecules

can become productive

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 8

22

With small aggregates,

Brownian movements are futile

ADP

Aggregate size may affect

a Brownian unfolding machine

23

To cooperate, individual Hsp70 (DnaK) molecules

do not need to touch each other directly; They may cooperate by sharing the same polypeptide substrate

The substrate

has multiple Hsp70

binding sites!

Ben-Zvi et al., JBC, 2004, 279: 37298-303

Dn

aK

refo

ldin

g a

cti

vit

y

DnaK (µM)

24

We would realise that the bound Hsp70

would rapidly collide with each other

What would happen if we took

into account the actual realistic molecular volumes in this drawing?

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 9

25

Another question:How may cooperati ve “tumbling” between several Hsp70 molecul es

bound to different unfolded segments of the same misfolded polypepti de,

lead to the unfolding of misfolded beta-sheet structures that must be,

by definition, in between the unfolded binding sites for Hsp70s?

26

Hsp70 is central to the active import of proteins in organelles

Nature Reviews Neuroscien

ce, 6

: 11-2

2 (2

00

5)

27

Post-transcriptional import into the mitochondria,

the problems:

1) Most of the mitochondrial proteins which are first synthesized

on cytoplasmic ribosomes are unstabl e in the unfolded state;

Therefore they must reach a native-li ke conformation,

or aggregate, already before import

2) The mitochondri al inner membr ane needs to keep

a strong proton gradient for ATP synthesis;

It cannot afford large pores to import already folded protein structures

1) The import pore must be as narrow as possible and gated;

It will allow the passage of only unfolded protein structures

2) To be imported, the near-nati ve cytopl asmic proteins, need to be

a) Targeted to the pore by way of a transit peptide

b) Locally unfolded to allow translocation but not globally unfolded

which would cause aggregation

The solution:

van der Laan et al., FEMS Yeast Res., 6: 849–861, 2006

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+

H+H+ H+

H+

H+

H+

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 10

28

Hsp70 as a translocase

Hsp70 would exert a pulling forceby way of a lever arm movement

that needs an anchor (Tim44)Proteins can arrive

at membranes in a compact form, bigger than the pore,

and need to be opened

The protein goes back and forth because

of thermal fluct. The role of Tim44 would be

to just target mtHsp70 at the pore exit and increase

the binding probability

Tim44 recruits mtHsp70 at the pore exit

Neupert & Brunner, Nat. Rev. Mol. Cell Biol., 3: 555, (2002)

Proteins spontaneously unfold, because of thermal fluctuations

(rates ~ 10-2-10-4 s-1)

Hsp70 is bulky and would simply block retro-translocation because it cannot enter the pore

A. Power-stroke model

B. Brownian-ratchet model

29

WT = wild typepreserved interaction

between Hsp70 and Tim44

ssc1-2 = mutants without interactions

between Hsp70 and Tim44 → no power stroke

Without anchor point for a lever-arm movementimport is preserved (Brownian ratchet)

but slower for folded proteins (Power-stroke)

Evidence for both

Brownian ratchet and power strokeWhich model correctly describes

the physical mechanism of action of Hsp70?

Lim et al., EMBO J., 2001, Mar. 1, 20(5): 941-50

30

Hsp70 as a disaggregating and reactivating chaperone:

evidence for an active unfolding action

Hsp70 as a translocation motor:

evidence for an active unfolding action

evidence for a ratchet mechanism

Summarizing

Need to find the source

for a force of unfolding

Need for a mechanism encompassi ng

both power-str oke and Brownian ratchet

In disaggregation and reactivation there are neither

pores for a Brownian ratchet, nor anchor points for a power-stroke

We must seek a new way to generate a force

and to rectify thermal fluctuations, unify ing the different functions

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 11

31

Two conflicting models:

Brownian ratchet versus power-stroke

Spontaneous unfoldingby Brownian ratchet

W alter Neupert

Hsp70 must be releasedfrom TIM44 during translocation

Active unfoldingby power-strokes

Jeff Schatz

Hsp70 must be anchoredto TIM44 during pulling

32

As soon as mtHsp70 binds and locks on to the incoming peptide,

it loses its high affinity for Tim44 and is allowed to diffuse away

from the membrane; This applies a pulling force of entropic origin

on the chaperone- bound imported polypeptide

Regardless of the presence of ATP or ADP,mtHsp70 binds Tim44 with high affinity

as long as it is not binding to an incoming peptide

6-66-66-66-

33-33-33-33

AMPADPATPNo nucl.

Tim

44

alo

ne

Hsp70 [µM]

P5 [µM]

Tim44-hsp70

Tim44

P5 - CALLLSAPRR

TIM44

J

ATP

No peptide

+ peptide

TIM44

J

Tim44 binding

and substrate binding

to mtHsp70

are mutuall y exclusi ve

Azem et al., in press

33De Los Rios et al., (2006), PNAS, 103: 6166-71

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 12

34

The entropic pulling region

Characterization of an entropic pulling zonenearby the import pore: a thermodynamic analysis

taking into account exclusion volumes of macromolecules

De Los Rios et al., (2006), PNAS, 103: 6166-71

35De Los Rios et al., (2006), PNAS, 103: 6166-71

36De Los Rios et al., (2006), PNAS, 103: 6166-71

Entropic pullingexplains why in Tim44 depleted ts mutants slower

protein import can still take place,

likely with the help of soluble Hsp40 (Mdj1)

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 13

37Nature Reviews Neuroscience, 6: 11-22, (2005)

Hsp70 disaggregates

and refolds misfolded proteins

38

In addition to pulling imported proteins,

mtHsp70 can disaggregate proteins

De Los Rios et al., (2006), PNAS, 103: 6166-71

This points to the J-domain protein

(DnaJ or PAM16/18 and/or PAM17)

as the principal Hsp70 entrapping device

How can Hsp70 pull on aggregates protein segments

in the absence of a TIM44 anchor and of a pore?

39

For Hsp70 to (reluctantly) enter the entropic pulling region -

in the first place, it needs energy

Where does this energy come from?

How can Hsp70 be entrapped in a place,

where its freedom is thermodynamically constrained?

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 14

40

Hsp40 (DnaJ) proteinshave at least two domains with two different functions:

thus, Hsp40 can serve as a “glue”

to entrap Hsp70 near the aggregate,

within the entropic pulling region

Triggers the locking of Hsp70 onto its misfolded substrate Binds to misfolded proteins

C-terminusCys repeats

41

Hsp40 entraps Hsp70 within the entropic pulling region

The aggregate is the sticky fly-paper

The Hsp70 is a blind fly

Hsp40 is the glue

Without “glue”, the fly (Hsp70) bounces without effect

The role of Hsp40:

to entrap Hsp70 within the entropic pulling region

With the DnaJ “glue”, Hsp70 can become entrapped

within the entropic pulling region

Force

42

The HSP70 cycle:

unfolding by entropic pullingForceful local unfolding for disaggregation and native refolding

DnaJ-binding

1

Misfolded segment

Substrate(aggregate)

J JDnaK-binding

2

ATP

DnaJ-acceleratedATP-hydrolysis,DnaK locking

3

J

ADP

Natively refolded segment

GrpE-acceleratedDnaK unlocking,spontaneous local

native refoldingProduct

(partially native)

5

Unfolded segment

4

Local unfoldingby entropic pulling,DnaJ-dissociation

ADP

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 15

43Sousa and Lafer, (2006), Traffic 8

90°

TD

Hsc 70

AuxilinTripod

Tail

TD Auxilin

44Nature Reviews Neuroscience, 6: 11-22, (2005)

Hsp70 disaggregates native Hsf1 trimers

and thus regulates its own expression

45

Thanks to:

Paolo De Los Rios Ecole Polytechnique Fédérale de LausanneThermodynamics

Anat Ben ZviHebrew Univ.NorthwesternBiochemistry

of DnaK

Ejal Gur Tel Aviv UnivMITRole of DnaJ

Abdulsalaam AzemTel Aviv Univ.mtHsp70

Fonds National Suisse

Schweizerischer Nationalfonds

Fondo Nazionale Svizzero

Swiss NationaL Science Foundati on

Towards a Unifying Mechanism

for the Hsp70 Chaperones

Prof. Pierre Goloubinoff

The screen versions of these slides have full details of copyright and acknowledgements 16

46