Chaperones involved in protein folding

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Chaperones involved in protein folding
Overview of molecular chaperone families -
distribution of chaperones in eukaryotes, archaea
and bacteria Nascent-chain binding chaperones -
Trigger Factor, NAC, Hsp70, prefoldin
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Overview of chaperone familiesDistribution
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Overview of chaperone familiesmultigene 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)
• must 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
aomtpkz------------------- GIM1 O COG1370
Prefoldin, molecular chaperone, alpha class
archaea
bacteria
yeast
other categories translation, transription, cell
motility, ion transport, etc. etc.
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Different sites of action
Location of chaperone is very important
cytosol? membrane? organelle?
extracellular? periplasmic?
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Co-localization

• 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 wasnt 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?

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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
co-translational 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
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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
salt-stripped 77aaffLuc RNCs (ribosome nascent
chains) obtained by in vitro translation in
rabbit reticulocyte lysate, and carrying the
photo-cross-linker (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.
77aaffLuc is the N-terminal 77 amino acids of
firefly luciferase lacking an import signal
Beatrix et al. (2000) J. Biol. Chem. 275, 37838.
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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 its not a prolyl isomerase?

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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
Hsp70s 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
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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

• Structure of entire molecule (70 kDa) has not
  been solved
• flexible linkage between ATPase and
  peptide-binding domains, and different
  conformations of molecule possible
• polypeptide-binding domain consists of
  beta-sheet scaffold loops possess hydrophobic
  residues that contact peptide
• domain also has an alpha-helical lid that is
  regulated by the ATPase activity

ATPase domain (homology with actin, which
also binds ATP)
Polypeptide binding domain with bound peptide
substrate
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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
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Hsp70 binds short hydrophobic sequences

• Binding sites are either completely buried or
  partially shielded
• Binding motif occurs every statistically
  occurs every 36 residues
• Consistent with general binding affinity for
  nascent polypeptide chains (estimated at 20 or
  more)

Rudiger et al. (1997) EMBO J. 16, 1501
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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 DnaKs 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 DnaKs 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

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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 Hsp70s, and some actually modulate
  the function or localization of Hsp70s
• There also exists a number of additional
  chaperone cofactors that modulate the activity of
  Hsp70s
• - e.g., Hip and Bag these affect ATPase
  activity of Hsp70
• in yeast, zuotin is an RNA-binding Hsp40
  chaperone that is ribosome-bound a cytosolic
  Hsp70 interacts with it to bind to nascent
  polypeptides

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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
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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

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Prefoldin functional mechanism (a)
PFD prefoldin Pa alpha subunit Pß beta
subunit
Siegert et al. (2000) Cell 103, 621.
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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

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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 dont have Hsp70, the
  otherwise ubiquitous molecular chaperone