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7-1
Chaperones involved in folding (I)
Overview of molecular chaperone families
- distribution of chaperones in eukaryotes, archaea and bacteria
Nascent-chain binding chaperones
- Trigger Factor, NAC, Hsp70, prefoldin
Overview of chaperone families:
Distribution
Eukaryotes
Archaea
NAC
NAC
Hsp70 system
[Hsp70 system]
prefoldin
prefoldin
chaperonins (group II) chaperonins (group II)
small Hsps
small Hsps
Hsp90
AAA ATPases
AAA ATPases
Hip, Hop, Bag, clusterin,
cofactors A-E, calnexin,
calreticulin, etc. etc.
Bacteria
Trigger Factor
Hsp70 system
chaperonins (Group I)
[small Hsps]
[Hsp90]
AAA ATPases
SecB
[PapD/FimC]
-
7-2
Overview of chaperone families:
multigene families
 not all molecular chaperone families are present in the three domains of life;
some are highly specialized and are found in just one domain
 eukaryotes have evolved not only more different families of chaperones, but
typically have more members (e.g., Hsp70, small Hsps, prefoldin, etc.)
 related to diversity of processes? (eukaryotes have organelles, greater diversity
of cell functions)
 can perform comparitive studies, e.g., with genome of the microsporidian
Encephalitozoon cuniculi, 2.9 Mb. Amitochondriate, parasitic; cause of severe
infections
 bacteria and archaea do have chaperone multigene families
 potential overlap in function? (e.g., Hsp70 in same/different compartments)
 replacement of function by other chaperone families (e.g., prefoldin)
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COG
“Clusters of Orthologous Groups of proteins”
Homologues: genes that are related in sequence and function
Orthologues: cross-species or cross-domain genes that are related in sequence and function
Paralogues: homologous genes that were duplicated in the same organism
http://www.ncbi.nlm.nih.gov/COG/xindex.html
category: O Post-translational modification, protein turnover, chaperones
* 15 --------qv--b-efghs-ujx-l-
HslU [O]
COG1220 ATP-dependent protease, ATPase subunit
* 48 aomtpkzy--drbc-f-----j----
SpoVK [O]
COG0464 ATPases of the AAA+ class
5 58 ---t---yqvdrbcefghsnujxilw
ClpA [O]
COG0542 ATPases with chaperone activity, ATP-binding domain
* 54 aomtpkzyqvdrbcefghsnujxilw
GroEL [O]
COG0459 Chaperonin GroEL (HSP60 family)
2 26 -------yqvdrbcefghsnujxilw
GroES [O]
COG0234 Co-chaperonin GroES (HSP10)
6 19 -------y---rbcefghs-ujx-l-
HtpG [O]
COG0326 Molecular chaperone, HSP90 family
* 70 -o-tp--yqvdrbcefghsnujxilw
DnaJ [O]
COG0484 Molecular chaperones (contain Zn finger domain)
7
-------------ce---s-uj----
CbpA [O]
COG2214 Molecular chaperones, DnaJ class
* 36 aomtpkzyqvdrbcefg-s---x---
IbpA [O]
COG0071 Molecular chaperone (small heat shock protein)
3 10 aomtpk-yq-----------------
GIM5 [O]
COG1730 Prefoldin, molecular chaperone, beta class
9
GIM1 [O]
COG1370 Prefoldin, molecular chaperone, alpha class
aomtpkz-------------------
archaea
bacteria
yeast
other categories: translation, transription, cell motility, ion transport, etc. etc.
7-5
Different sites of action
Location of chaperone is very important:
cytosol?
membrane?
organelle?
extracellular?
 e.g., calnexin; must be
near polypeptide entry?
 ribosome-bound?
 soluble?
 associated with
particular structures?
 must bear sequence tag
to target it there
 chaperonin required
for its own folding
periplasmic?
 e.g., clusterin
binds large number
of extracellular
proteins
 e.g., PapD/FimC is
required for pilus
folding/assembly
Co-localization / aggresomes
chaperones can co-localize with:
 other chaperones
 protein degradation machinery
 different substrates
 etc.
Example:
- misfolded proteins may end up in aggresomes
(e.g., CFTR)
- aggresomes contain various molecular
chaperones, including Hsp70 and Hsp40, as well
as proteasome components
This can potentially cause problems:
- researchers expressed mutant CFTR
- they then expressed mutant GFP that is normally broken down
- saw GFP fluorescence (green) in the cytosol (i.e., it wasn’t degraded)
- has implications for proteins that aggregate in cell and cause diseases
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Nascent-chain binding chaperone: TF
Trigger Factor (TF)
- most effective peptidyl prolyl isomerase (PPIase)
- behaves as a conventional molecular chaperone, i.e., can bind non-native proteins
- ribosome-bound (interacts with RNA in the 50S ribosome subunit, but some of it is
cytosolic)
- interacts with large fraction of nascent polypeptides (as determined by cross-linking)
- only occurs bacteria (where it is ubiquitous), although other eukaryal/archaeal
proteins have FKBP domains
- deletion is not lethal(!) However, deletion is lethal when knock out bacterial Hsp70,
which also binds nascent chains
-crystal structure suggests that it forms a ‘pocket’ for chains exiting the ribosome
(recall the ‘crouching Dragon’ structure presented in class)
• how do the chaperone binding site and PPIase cooperate?
• what is the exact nature of the polypeptide binding site?
TF bound to ribosome
7-8
Baram et al. PNAS 2005
Nascent-chain binding chaperone: NAC
Nascent polypeptide Associated Complex (NAC)
- eukaryotic protein consists of alpha and beta subunits; archaea have only beta subunit
- as with TF, bound to ribosome
- does not contain domain resembling a PPIase
Primary function:
- prevents inappropriate targeting
of nascent polypeptides by SRP
- if ER signal sequence is present,
SRP binds it, causes translation
arrest, and docking occurs; cotranslational insertion of protein
then takes place, and the
sequence is cleaved
- if ER sequence is not present,
NAC prevents SRP from binding
to the nascent chain
- evidence suggests it may help
targeting to mitochondria
7-9
NAC function: example experiment
Fig. 8. NAC complex, but not the individual
subunits, prevent inappropriate interaction of SRP
with signal-less chains on ribosomes. High saltstripped 77aaffLuc RNCs (ribosome nascent
chains) obtained by in vitro translation in rabbit
reticulocyte lysate, and carrying the photo-crosslinker (TBDA-modified lysine-tRNA), were
incubated first with excess SRP, then with the
individual NAC subunits, bovine NAC, or
recombinant NAC as indicated. Samples were
irradiated and analyzed by SDS-PAGE and
fluorography. Bovine NAC (lane 6) and the
reconstituted recombinant NAC (lane 5) both
successfully competed with SRP for interaction
with a signal-less chain on the ribosome. But
neither alpha-NAC (lane 3) nor beta-NAC (lane 4)
alone could prevent SRP from interacting with the
signal-less nascent chain on the ribosome.
1. translate 77aaffLuc in RRL in presence of TBDA-Lys-tRNA and
SRP/NAC components
2. photoactivate cross-linker
3. look for cross-linking between SRP, alpha/beta NAC and aaffLuc
Beatrix et al. (2000) J. Biol. Chem. 275, 37838.
77aaffLuc is the N-terminal 77 amino
acids of firefly luciferase lacking an
import signal
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NAC: a bona fide chaperone?
 If NAC is present at the polypeptide exit tunnel,
and generally binds nascent chains (except when it is
displaced by SRP), could it act as a molecular
chaperone?
 Is NAC functionally equivalent to Trigger Factor
except for the fact it’s not a prolyl isomerase?
7-12
Nascent-chain binding chaperone: Hsp70
Found in nearly all compartments where protein folding takes place:
- cytosol of eukaryotes (Hsp70) and bacteria (DnaK)
- mitochondria (mt-Hsp70)
- chloroplast (cp-Hsp70)
- endoplasmic reticulum (BiP)
- in yeast and nematodes, there are at least 14 different Hsp70’s
One surprising exception:
- not found in all archaea; this has been viewed as a paradox
- reason is that it has been shown to bind nascent polypeptides:
- it can be cross-linked to nascent chains in eukaryotes and bacteria
- another reason is that it is important for de novo protein folding
Hsp70 in de novo protein biogenesis
 Hsp70 is believed to bind and stabilize nascent polypeptides early in their
synthesis--preventing misfolding and aggregation
 Hsp70 binding and release, in an ATP-dependent manner, may help proteins fold to
the native state OR Hsp70 may ‘transfer’ non-native proteins to other chaperones for
folding (e.g., chaperonins)
 Hsp70 is also important during cellular stresses (thermotolerance), and has
numerous other functions in the cell apart from assisting de novo protein folding. It
often works in collaboration with other chaperones, especially Hsp40
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Structure of Hsp70 chaperone
 flexible linkage between
ATPase and peptide-binding
domains, and different
conformations of molecule
possible
Polypeptide binding domain
with bound peptide
‘substrate’
 polypeptide-binding domain
consists of beta-sheet scaffold;
loops possess hydrophobic
residues that contact peptide
 domain also has an alphahelical ‘lid’ that is regulated by
the ATPase activity
Jiang et al. (2005) Mol. Cell 20, 513-24.
Structural Basis of Interdomain
Communication in the Hsc70 Chaperone
7-15
Substrate specificity of Hsp70
Experiment
1. synthesize 13-mer peptides that overlap by 10 amino acids,
based on actual protein sequences (spacer is Ala2)
- this covers entire protein sequence and any binding site
2. cross-link peptides to nitrocellulose membrane (automated)
3. add chaperone and allow binding to equilibrium
4. electro-transfer any Hsp70 bound to peptides onto membrane
5. probe membrane by Western blotting with specific antibody
6. screen 37 different proteins this way
7. obtain statistically significant information on binding motif
citrate synthase (full length)
1
2
3
covalently
linked to
membrane
12 3
nitrocellulose
peptides
incubate with chaperone
(protein of interest)
transfer to other
membrane and perform
Western Blot
see which peptides the
protein binds
Hsp70 binds short hydrophobic
sequences
7-16
alkaline phosphatase
catabolite activator protein
influenza hemagglutinin
 Binding sites are either completely buried or partially shielded
tumour suppressor
Rudiger et al. (1997) EMBO J. 16, 1501
Binding “ motif ” occurs every statistically occurs every 36
residues
 Consistent with general binding affinity for nascent
polypeptide chains (estimated at 20% or more)
7-17
Bacterial DnaK functional cycle
 DnaJ (Hsp40 homologue) has
affinity for unfolded proteins, and can
deliver a substrate to DnaK
 DnaK has fast on- and off-peptide
binding rate when ATP is bound
 DnaJ helps accelerate DnaK’s
ATPase
 DnaK has slow on- and off-peptide
binding rate when in ADP
conformation (i.e., it binds stably)
 GrpE is a nucleotide exchange
factor; it ‘opens’ up DnaK’s
nucleotide binding site to help it
release ADP and re-bind ATP
 Released proteins may then be
folded or might re-bind DnaJ/DnaK
for another round of folding, or may
interact with a chaperonin
7-18
DnaJ (Hsp40)
 Hsp40 may bind nascent polypeptides directly, passing these on to Hsp70
 although it is a molecular chaperone in its own right, it seems to operate
mostly in conjunction with Hsp70
 there are numerous Hsp40 homologues in eukaryotes and bacteria; some
are specific for the different Hsp70’s, and some actually modulate the
function or localization of Hsp70’s
 There also exists a number of additional chaperone cofactors that
modulate the activity of Hsp70’s:
- e.g., Hip and Bag; these affect ATPase activity of Hsp70
 in yeast, zuotin is an RNA-binding Hsp40 chaperone that is ribosomebound; a cytosolic Hsp70 interacts with it to bind to nascent polypeptides
Nascent-chain binding chaperone:
prefoldin
Discovery
- a group performed a screen for yeast genes that were synthetically lethal in
combination with a gamma-tubulin mutation
- found 5 genes that when disrupted, resulted in cytoskeleton defects
• actin: sensitivity to osmotic stress, latrunculin-A; disrupted actin filaments
• tubulin: sensitivity to benomyl; disrupted microtubules
- another lab independently purified a bovine protein complex containing 6 proteins
that could bind unfolded actin and tubulin; the yeast complex was later purified and
shown to possess the same 6 orthologous proteins as the bovine complex
Characterization
- synthetic lethality with various actin and tubulin mutants, as well as mutants
involved in microtubule processes (i.e., cofactors A-E)
- may cooperate with cytosolic chaperonin (CCT) in actin and tubulin biogenesis
7-19
Prefoldin subunit structure
Predicting coiled coils in proteins:
- a number of web-based programs are available
- rely on the repeating unit of the coiled coil
- a and d positions in a-g heptad repeat are usually
hydrophobic
- the a and d positions form the apolar interface between
the two helices; because of alpha helices normally have 3.6
residues/turn, the 3.5 residues/turn of the coiled coil
induces a strain on the helix
Some coiled coils can have
three or more helices
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Prefoldin quaternary structure
 most of surface is
hydrophilic in character
 inside tips of the coiled
coils and ‘bottom’ of
cavity display some
hydrophobic character
 Structure of archaeal
prefoldin hexamer
 oligomerization domain
is a double beta-barrel
structure
 coiled coils are ~80A
long and would be
expected to behave
independently
7-22
Prefoldin functional mechanism (a)
PFD = prefoldin
Pα = alpha subunit
Pβ = beta subunit
Siegert et al. (2000) Cell 103, 621.
7-23
Prefoldin functional mechanism (b)
 Binding of prefoldin to
unfolded proteins requires the
multivalent interaction of the
coiled coils
 many other chaperones also
bind in a multivalent manner
Prefoldin functional mechanism (c)
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Hsp70-like function of prefoldin?
 Prefoldin is found in all archaea but Hsp70 is not; those that have Hsp70 probably
acquired it via lateral gene transfer
 Mechanism of prefoldin is clearly different from that of Hsp70, but the overall
function of each may be similar:
- both bind nascent polypeptides
- prefoldin can stabilize an unfolded protein for subsequent folding by chaperonin
(explanation in class)
- range of proteins archaeal prefoldin stabilizes is considerable: 14-62 kDa
 Archaeal prefoldin (with 2 different subunits) may play a general role in protein
folding whereas the eukaryotic chaperone (with 6 different subunits) may have
acquired more specialized functions; this is seemingly the case for the eukaryotic
chaperonin CCT, which has 8 different subunits compared to the archaeal chaperonin,
which has 1 or 2 subunits, and the bacterial chaperonin (GroEL), which has 1 subunit
 the presence of prefoldin may resolve the paradox that many archaea don’t have
Hsp70, the otherwise ubiquitous molecular chaperone