Protein folding in the cell

1
Protein folding in the cell
Basics - cell compartments, molecular crowding
cytosol, ER, etc. Folding on the ribosome -
co-translational protein folding Molecular
chaperones - concepts, introduction -
intramolecular chaperones - chemical chaperones -
protein chaperones
2
Cell compartments and folding
eukaryotes - cytosol ..........................
........protein synthesis, folding/assembly -
extracellular .........................proteins
are exported in folded form - mitochondria
........................limited protein
synthesis energy production - chloroplasts
..........................limited protein
synthesis light harvesting - endoplasmic
reticulum.......... import of unfolded proteins
protein processing - peroxisome
........................... import of folded
proteins anab./catab. pathways - nucleus
................................. import of
folded proteins - lysosome.......................
......... import of unfolded proteins degradation
bacteria - cytosol ............................
......protein synthesis, etc. - periplasm
.............................import and folding
of periplasmic proteins - extracellular
.........................proteins are exported
archaea - cytosol ...............................
...protein synthesis, etc. - extracellular
.........................proteins are exported
3
Folding in vitro vs. in vivo
in vitro
in vivo
protein denatured in a chaotrope
Differences 1. One has all of the information
immediately available for folding the other
process is gradual 2. the cellular environment is
very different (much more crowded)
folding by dilution in buffer
folding
folded protein
folded protein
4
Co-translational protein folding
Fact - first 30 amino acids of the polypeptide
chain present within the ribosome is
constrained (the N-terminus emerges
first) Assumption as soon as the nascent chain
is extruded, it will start to fold
co-translationally (i.e., acquire secondary
structures, super-secondary structures, domains)
until the complete polypeptide is produced and
extruded
folding
assembly
5
Sindbis Virus Capsid Protein (SCP)
SCP is the capsid protein of the Sindbis
virus 26S Sindbis RNA encodes a polyprotein
SCP is auto-proteotically cleaved from the rest
of the polyprotein other cellular proteases
cleave E1-E3 from the polyprotein to generate the
mature proteins E1, the envelope protein, is 9
kDa SCP is a 33 kDa serine protease WT SCP
self-cleaves Ser215 gt Ala215 mutant doesnt
N
C
SCP
E1
E2
E3
6
SCP folds co-translationally
Experiment 1. make and translate different SCP
construct RNAs in vitro in the presence of
35S-methionine for 2 min 2. Prevent re-initiation
of translation with aurintricarboxylic acid
(ATCA) synchronizing 3. at set timepoints, add
SDS buffer and perform SDS-PAGE 4. observe by
autoradiography

N
C
SCP
E1
N
C
SCP
N
C
SCP
E1
7
Macromolecular crowding
in vitro
lt0.1 mg/ml
Ellis and Hartl (1996) FASEB J. 1020-26
When doing experiments in vitro, we should all be
thinking about thisproteins in isolated (pure)
systems may not behave as they do in the cell-
binding partner(s) might be missing - cell
conditions (pH, salts, etc.- post-translational
modifications might be missing may be
dramatically different
8
Effects of crowding
Definition Molecular crowding is a generic term
for the condition where a significant volume of a
solution, or cytoplasm for example, is occupied
with things other than water Fact - association
constants (ka) increase significantly -
dissociation constants (kd) decrease
significantly (kd1/ka) - increased on-rates
for protein-protein interactions (see for
example Rohwer et al. (2000) J. Biol. Chem. 275,
34909) Assumption - non-native polypeptides
will have greater tendency to associate
intermolecularly, enhancing the propensity of
aggregation
9
Effects of crowding example
denatured lysozyme, reduced or oxidized
dilution in buffer with different crowding agents
loss of activity due to protein aggregation
measure lysozyme activity
crowding agents ficoll 70, dextran 70, protein
(BSA, ovalbumin)
roughly spherical polysaccharide
van den Berg et al. (1999) EMBO J. 18, 6927.
10
Problem non-native proteins
non-native proteins expose hydrophobic residues
that are normally buried within the core of the
protein these hydrophobic amino acids have a
strong tendency to interact with other
hydrophobic (apolar) residues - especially
under crowding conditions
11
Solution molecular chaperones
12
Molecular chaperonesgeneral concepts
Requirements for a protein to be considered a
chaperone (1) interacts with and stabilizes
non-native forms of protein(s) -
technically also folded forms that adopt
different protein conformations (2) not part of
the final assembly of protein(s) Functions of a
chaperone classical - assist folding and
assembly more recent - modulation of
conformation - transport - disaggregation of
protein aggregates - unfolding of proteins
self-assembly refers to the folding of the
polypeptide, as well as to its assembly into
functional homo- or hetero-oligomeric structures
13
Molecular chaperonescommon functional assays
Type of assay Rationale
Binary complex formation If chaperone has high enough affinity for an unfolded polypeptide, it will form a complex detectable by co-migration by SEC co-migration by native gel electrophoresis co-immunoprecipitation
Prevention of aggregation Binding of chaperones to non-native proteins often reduces or eliminates their tendency to aggregate. Assay may detect weaker interactions than is possible with SEC
Refolding Chaperones stabilize non-native proteins some can assist the refolding of the proteins to their native state. Usually, chaperones that assist refolding are ATP-dependent
Assembly Some chaperones assist protein complex assembly
Activity Some chaperones modulate the conformation/activity of proteins
(Miscellaneous) A number of chaperones have specialized functions
14
Intramolecular chaperones
Concept - portions of a polypeptide may assist
the biogenesis of the mature protein without
being part of the final folded structure - these
regions are chaperones by definition, although
classical molecular chaperones act
inter-molecularly, not intra-molecularly.
15
Intramolecular chaperone example
Subtilisin E - non-specific protease - mature
protein cannot fold properly if propeptide is
removed
propeptide (77 aa)
precursor (352 aa)
mature protein (275 aa)
Shinde et al. (1993) PNAS 90, 6924.
16
Intramolecular chaperone continued
Subtilisin E propeptide - unstructured alone in
solution - alpha-helical when complexed with
subtilisin? propeptide is 20 of preprotein CD
suggests combination mature subtilisin
propeptide mostly helical
propeptide with subtilisin
propeptide in TFE
propeptide
ellipticity
subtilisin
nm
NoteCD traces are additive

• Propeptide must interact with subtilisin

17
Intramolecular cleavage or intermolecular?
Fact unfolded His10-preprotein can refold alone
in solution
Experiment 1. prepare subtilisin pre-protein
containing an N-terminal polyhistidine tag
(His10) 2. unfold in denaturant 3. bind different
concentrations of the protein to Ni2-NTA
resin 4. assay for folding by measuring
propeptide release
Q what do the results mean? Q why bind the
protein to a resin? Q why use different
concentrations of proteins?
Li et al. (1996) J. Mol. Biol. 262, 591.
18
Chemical chaperones
Concept - small molecules could enhance the
stability and assist the folding or assembly of
proteins - under conditions of cellular stress,
such as a heat-shock, small molecules may help
proteins from misfolding and aggregating - one
easy way to test is to see how they can prevent
loss of activity, or, prevent the aggregation of
a protein - protein aggregation can be
conveniently monitored spectrophotometrically
at 360 nm, where light scattering from the
aggregates is detected
19
Chemical chaperones example
A
B
protein aggregation
Singer and Lindquist (1998) Mol. Cell 1, 639.
20
Chemical chaperones example
B
C
40ºC heat shock
40ºC heat shock
Note tps1 yeast cells have a deletion in the
trehalose synthase
21
Different chemical chaperones
without
with
protein aggregation
protein aggregation
22
trans-acting protein molecular chaperones
- cis-acting (intramolecular) chaperones are
relatively rare - chemical chaperones may play an
important role in protecting proteins in the
cell, but their extent of action is likely to be
limited - organisms have evolved large families
of protein molecular chaperones that have either
general functions in the cell, or have highly
specific functions - the expression of many of
the chaperones is induced under cellular stress
conditions--giving rise to the name Heat-shock
proteins, or Hsps, followed by their Molecular
Weight (MW) BUT - not all chaperones are
Hsps - not all Hsps are chaperones
23
Molten globules
- intermediate conformation assumed by many
globular proteins under mildly denaturing
conditions
- as determined experimentally using experimental
techniques involving hydrogen exchange,
small-angle X-ray Scattering (SAXS), circular
dichroism, fluorescence spectroscopy, binding of
hydrophobic probes, etc.
characteristics
presence of substantial content of secondary
structure absence of most of the specific
tertiary structure associated with tight packing
of side-chains dynamic features of the
structure with motions on a timescale longer than
nanoseconds the protein is still compact, but
radius is 10-30 larger compared with that of
native state presence of loosely-packed
hydrophobic core greater exposure of apolar
side-chains
24
Lysozyme molten globule
molten globule states notice how two different
folding paths converge into a local minimum
(lower energy state) that is close to, but has
not reached, the lowest energy (folded) state
Similar molten globule states very likely occur
in vivo
Native state
25
Molten globules inside the cell
Molten globules might be found - during
co-translational protein folding - before
translocation into a membrane - following
extrusion through a membrane - following
cellular stress
26
Probing protein structure with ANS
1-anilinonaphthalene-8-sulfonic acid
Excitation wavelength (maximum at 370 nm)
Emission wavelength (maximum at 480 nm)
- Fluorescence emission maximum and strength
changes upon binding to a hydrophobic region(s)
of proteins
27
Bis-ANS
4,4'-dianilino-1,1'-binaphthyl- 5,5'-disulfonic
acid
- strength of emission is better than ANS -
photoincorporation occurs with UV irradiation
- retains fluorescence when covalently bound
28
Background for two papers
Chaperonins. Large, double-ring structures that
accept non-native proteins inside their cavities
and assist protein folding. The eukaryotic
cytosolic chaperonin is involved in folding
actins and tubulins. Prefoldin. Hexameric
molecular chaperone also involved in actin and
tubulin biogenesis. Its existence was not known
when the Cell paper was published in 1997 (it was
discovered in 1998). It is also known as Gim
complex, or GimC. Stochastic model for de novo
protein folding. The definition of stochastic is
involving or containing random variables. In this
context, it means that folding polypeptides will
interact with whichever molecular chaperone may
be present at any one time during its synthesis.
If the protein has not folded or assembled after
interaction with chaperone(s), it will be
released in the bulk cytosol and may interact
with other chaperone(s) before it has a chance to
fold/assemble. Pathway model for de novo protein
folding. Hypothesis that many newly-synthesized
cellular proteins follow an ordered pathway
during de novo protein folding. This ordered
pathway would typically occur when one or more
molecular chaperones interact with the newly made
protein.
BUT NOTE THAT MOST PROTEINS can probably fold in
vivo either with minimal or no assistance from
molecular chaperones!!!
29
Protein folding catalysts
Protein folding catalysts - peptidyl prolyl
isomerases (PPIases) - protein disulfide
isomerases (PDIases)
30
The proline peptide bond
- proline, an imino acid
180º
trans conformation favoured 1000-fold over
cis Rside chain OC-N-H is planar
trans 93
cis 7
- cis conformation rare except for proline - cis
can be 10-30, depending on the nature of the
Xaa-Pro bond
31
Proline cis-trans isomerization
- slow because it involves rotation about a
partial double-bond (t1/2 between 10-100 sec at
25ºC) - cis-trans equilibria more common in
flexible regions of native proteins (e.g.,
coils)OR during protein folding - strong acids
favour cis-trans isomerization by protonating the
nitrogen atom - proline residues disrupt
alpha-helices often found in turns - cis-trans
isomerization could be used as a molecular switch
H-
Catalysis of cis-trans isomerization - simple
reaction does not involve breaking or forming
bonds - mechanism catalysis by distortion and
transition state containing partially-rotated C-N
bond - this would result in a reduced partial
double-bond character PPIase, Peptidyl Prolyl
Isomerase, catalyzes proline cis-trans
isomerization - active site of PPIase hydrophobic
in character conserved Arg residue of a PPI
might be involved in H-bond formation with N,
producing C-N bond with more single-bond character
H-
32
Peptidyl prolyl isomerases
Three classes are known
Cyclophilins - ubiquitous 11 different ones
found in S. cerevisiae not essential for
viability - binds cyclosporin A
FKBP binding proteins- no sequence similarity
with cyclophilins many different members found
in eukaryotes, as well as prokaryotes not
essential for viability. Yeast mutant lacking all
its cyclophilins and FKBP binding proteins still
alive!- bind immunosuppressants FK506 and
rapamycin (but not cyclosporin A) - both
cyclophilins and FKBPs form complexes with the
molecular chaperone Hsp90, perhaps to catalyze
cis-trans isomerization as well as to assist
folding (or modulate protein conformation)
Parvulins- not related to cyclophilins or FKBP
binding proteins, and are not inhibited by
cyclosporin A, FK506 or rapamycin- occur as
small proteins of lt100 amino acids or as domains
of larger proteins- have high PPIase activity
33
Assay methods for PPIases
Chymotrypsin-coupled assay - chymotrypsin cleaves
only the trans-form of the Xaa-Pro bond amino
acid of a small model peptide such as
N-succinyl-Ala-Xaa-Pro-Phe-p-nitroanilide - in
aqueous solution, 90 of Xaa-Pro bond of this
molecule is in trans-conformation - after
addition of excess amount of chymotrypsin, the
trans form of Xaa-Pro bond is cleaved
instantaneously - hydrolysis rate of the
remaining 10 Xaa-Pro bond is limited by its cis
to trans isomerization - cis-trans isomerization
rate of model peptide is measured by the release
of p-nitroanilide spectrophotometrically
Chymotrypsin-free assay- in a mixture of TFE and
LiCl, the N-succinyl-AXPF-p-nitroanilide peptide
is approximately 50 in the cis conformation-
upon dilution in buffer, cis-trans isomerization
occurs, decreasing cis content to 10- small
differences in absorbance between the cis and
trans forms of the prolyl imide bond in the model
peptide are then measured at 330 nm
NMR- could also monitor cis-trans isomerization
by nuclear magnetic resonance (NMR), but this
method is more expensive, slower, and requires
more protein
34
Cyclophilins
Discovery - 1989 an 18 kDa, cytosolic peptidyl
prolyl isomerase from kidney was shown to be
nearly identical to a cyclophilin known as Cyp A,
a receptor protein for the immunosuppressant
cyclosporin A - mechanism catalysis by
distortion and transition state containing
partially-rotated C-N bond - this would result
in a reduced partial double-bond character -
active site of PPIase hydrophobic in character
not known whether conserved Arg residue of a PPI
might be involved in H-bond formation with N,
producing C-N bond with more single-bond character
35
Cyclophilin A with Gly-Pro
Catalysis of cis-trans isomerization - simple
reaction does not involve breaking or forming
bonds - mechanism catalysis by distortion and
transition state containing partially-rotated C-N
bond - this would result in a reduced partial
double-bond character - active site of Cyp A
PPIase hydrophobic in character not known
whether conserved Arg residue of a PPI might be
involved in H-bond formation with N, producing
C-N bond with more single-bond character - The
pipecolic amide moiety of FK506, which probably
mimics the proline residue of peptide or protein
substrates, is bound in a hydrophobic pocket of
FKBP, presumably at the active site
cyclophilin A in complex with dipeptide, GP -
R55A mutant has lt0.1 activity
FK506
rapamycin
36
Chaperones involved in folding
Overview of molecular chaperone families -
distribution of chaperones in eukaryotes, archaea
and bacteria Nascent-chain binding chaperones -
Trigger Factor, NAC, Hsp70, prefoldin
37
Overview of chaperone familiesDistribution
Eukaryotes Archaea Bacteria
- - Trigger Factor
NAC NAC -
Hsp70 system Hsp70 system Hsp70 system
prefoldin prefoldin -
chaperonins (group II) chaperonins (group II) chaperonins (Group I)
small Hsps small Hsps small Hsps
Hsp90 - Hsp90
AAA ATPases AAA ATPases AAA ATPases
- - SecB
- - PapD/FimC
Hip, Hop, Bag, clusterin, cofactors A-E, calnexin, calreticulin, etc. etc. - -
38
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)

39
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.
40
Different sites of action
Location of chaperone is very important
cytosol? membrane? organelle?
extracellular? periplasmic?
41
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 wasnt degraded) - has
implications for proteins that aggregate in cell
and cause diseases
42
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?

43
TF bound to ribosome
Baram et al. PNAS 2005
44
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
45
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
46
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

47
Structure of Hsp70 chaperone

• 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

Polypeptide binding domain with bound peptide
substrate
ATPase domain (homology with actin, which
also binds ATP)
Jiang et al. (2005) Mol. Cell 20,
513-24. Structural Basis of Interdomain
Communication in the Hsc70 Chaperone
48
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
49
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
50
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
51
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
52
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

53
Prefoldin functional mechanism (a)
PFD prefoldin Pa alpha subunit Pß beta
subunit
Siegert et al. (2000) Cell 103, 621.
54
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

55
Prefoldin functional mechanism (c)
56
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

57
GroEL/GroES chaperonin system

• GroEL forms homo-oligomeric toroidal complex
  dependent on GroES cofactor for function GroEL
  is essential for cell viability
• GroEL/GroES system may bind 10 of all bacterial
  cytosolic proteins but recent study shows
  only a portion of those are completely
  chaperonin-dependent
• Belongs to so-called Group I chaperonins which
  includes evolutionarily-related bacterial GroEL,
  mitochondrial Hsp60, and chloroplast Rubisco
  subunit-binding protein (Rubisco is most abundant
  protein on earth and requires chaperonin for
  folding)
• Functional mechanism is the best understood of
  all chaperonins

58
GroEL/GroES structure
crystal structure of E. coli GroEL/GroES

• GroEL has two stacked heptameric rings
  (equatorial domains form inter-ring contacts)
• GroES forms a single heptameric ring that binds
  co-axially to one GroEL ring (caps GroEL,
  preventing polypeptide exit or entry) binds only
  when GroEL in ATP state
• crystals structure without GroES has been
  solved, and with ATP-gamma S (non-hydrolyzable
  ATP analogue)
• mitochondrial chaperonin (Hsp60) is single-ring
  GroES from chloroplasts consists of a fused dimer

59
GroEL subunit structure

• chaperonins have 3 domains
• equatorial domain is the ATPase
• intermediate domain is a flexible hinge binding
  of ATP and GroES causes the apical domain to move
  upward and turn about 90 to the side
• apical domain is the polypeptide binding domain
  the binding site consists mostly of large, bulky
  hydrophobic residues
• (determined by mutation analysis)
• GroES binds to the polypeptide binding site
  displaces substrate into the cavity

60
Group I chaperoninfunctional cycle

• large conformational changes occur upon ATP and
  GroES binding cavity interior expands 2 fold,
  hydrophobic residues in apical domain turn away
  from the binding site and the interior becomes
  hydrophilic
• ATP --gt ADP transition is when folding takes
  place in the cavity when ATP is hydrolyzed, and
  ATP/GroES binds to trans ring (opposite the cis
  ring), GroES on cis ring dissociates and the
  polypeptide exits
• the polypeptide may not be folded upon exiting
  it could undergo another round of folding by
  either the same chaperonin, another chaperonin,
  or another chaperone

61
GroEL mechanism of action
1. Multivalent binding of substrate 2. Unfolding
of substrate (controversial) - evidence that
non-native protein is unfolded further upon
binding to GroEL and hydrolysis of ATP 3.
Combination of multivalent binding, unfolding may
re-direct folding intermediates to proper folding
pathway once inside hydrophilic chaperonin
cavity 4. Infinite dilution??? (cage model)
Paper presentation (next 3 slides) Farr et al.
(2000) Multivalent binding of nonnative substrate
proteins by the chaperonin GroEL. Cell 100,
561-573.
62
GroEL function single polypeptide

• N- and C-termini of GroEL (chaperonins in
  general) are buried inside the cavity
• construct is a fusion between all 7
  subunits--protein size is 400 kDa!
• the fusion protein assembles properly as judged
  by em reconstructions
• powerful tool for analyzing contribution of
  individual subunits to binding, etc.

63
GroEL function in vivo

• strain with wild-type GroEL under control of lac
  promoter (inducible with IPTG)
• without IPTG, strain growth arrests
• growth restored when covalent GroEL (fusion
  construct) is present this represents a growth
  of
• other constructs were tested in the absence of
  IPTG o represents no growth, represents
  very slow growth

64
GroEL function in vitro

• found that covalent GroEL was a bit less active
  at binding non-native proteins compared to
  wild-type GroEL mild protease treatment restored
  binding
• experiment binding of denatured protein to
  various constructs, isolation by SEC, and amount
  of bound proteins quantitated

conclusions gt require at least two or three
GroEL subunits for binding non-native proteins
these should preferably be in positions 1-3 or
1-4 (i.e., not immediately adjacent) gt
ability of GroEL/GroES to fold substrate
followed similar pattern (not shown)
65
Group II chaperonin system
66
Group II chaperonin structure
alpha-helical protrusion
GroES
side view of top ring
apical domain
apical domain
side view of bottom ring
intermediate domain
intermediate domain
thermosome side view
equatorial domain
equatorial domain
GroEL
thermosome
comparison of GroEL/ES complex (one subunit of
GroEL, one subunit of GroES) with single
thermosome (alpha) subunit
8 subunits per ring 4 alpha, 4 beta subunits
thermosome top view
67
Group II chaperoninfunctional cycle

• open or closed states of thermosome (archaeal
  chaperonin related to CCT) were determined by
  SAXS experiments in the presence of nucleotides
  (ADP, ATP) or ADP in the presence of inorganic
  phosphate (PO4, or Pi) to simulate ADPPi
  transition state
• none of the studies have been carried out in
  presence of substrates assume open
  conformations can interact with substrate and
  closed state is involved in folding
• ATP?ADP transition somehow causes large
  conformational change

68
CCT-actin em reconstruction

• actin is composed of 4 subdomains, Sub1-Sub4
• hinge between domains Sub3-Sub4 and Sub1-Sub2 is
  flexible
• ATP binds in cleft between large and small
  domains
• actin cannot fold properly in the absense of ATP
• CCT-tubulin reconstruction also done tubulin
  makes more contacts with CCT subunits

69
Evolution of chaperonins, prefoldin and
actin/tubulin

• FtsA, actin homologue
• FtsZ, tubulin homologue

Evolution of eukaryotes

• CCT and prefoldin co-evolved essential for
  actin/tubulin biogenesis
• actin and tubulin are essential components of
  cytoskeleton
• cytoskeleton is required for large number of
  cell processes unique to eukaryotes, including
  intracellular movements, engulfment, etc. etc.
• hypothesis eukaryotes could not have evolved
  without CCT and prefoldin

70
Small heat-shock proteins

• found in all three domains of life, usually in
  multiple copies
• form large molecular weight complexes
• consist of three distinct domains

• can efficiently bind proteins on the aggregation
  pathway
• play important role in thermotolerance
  protecting proteins from aggregating under stress
  conditions
• cooperate with other chaperones (e.g., Hsp70) to
  renature proteins function, like that of
  prefoldin, is ATP-independent

71
Small Hsp crystal structure
- sizes of small Hsps range from 150 kDa to 800
kDa - smallest functional small Hsp is a
nonamer (trimer of trimer)

• crystal structure from Methanococcus jannaschii
  Hsp16 small Hsp (first archaeal genome to be
  sequenced) (wheat and ? Structures now also
  known)
• spherical shell composed of 24 subunits
• 2-, 3-, and 4-fold symmetry
• N-terminal domain (first 33 amino acids) were
  not resolved in the crystal structure these are
  likely to be flexible or disordered

72
Small Hsp surface view

• immunoglobulin domain fold (same as PapD/ FimC)
• dimer interface most extensive (building block)
• C-terminal region is exposed on surface
• N-terminal region faces interior of the oligomer
  (N-terminal region was not resolved in the
  crystal structure)

73
Wheat small HSP
End view
Side view
Dodecameric structure
van Montfort et al. Nature Structural Biology
(2001)
74
Hsp33 the redox chaperone

• exclusively bacterial induced during oxidizing
  (stress) conditions in the cell

Hsp33
oxidizing conditions (e.g., H2O2)
Hsp33/Hsp33 dimer
Hsp33

• domain-swapped dimer (active form) inactive
  monomer
• activation dependent on redox condition in cell
  under oxidizing (stress) conditions, disulfide
  bridges are formed and dimerization takes place
  conserved cysteines
• Hsp33 efficient in preventing protein
  aggregation in vitro

Jakob et al. (1999) Cell 96, 341.
75
Study questions

• What is the main difference between in vitro and
  in vivo protein folding?
• What do you understand by molecular crowding?
• What are the effects of molecular crowding?
• What is a chaperone?
• What is the definition of a chaperone?
• Explainn the action of a chaperone?
• What is Gro?
• Wha is its function?
• Why is IPP important?