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