Download lecture 8

8-1
Chaperones involved in folding (II)
Post-nascent-chain binding chaperones
- Chaperonins (bacterial GroEL, eukaryotic CCT, archaeal thermosome)
- Small heat-shock proteins (Hsps)
- Hsp33
8-2
A chaperone for a-hemoglobin
alpha-hemoglobin stabilizing protein (AHSP)
a
alpha-beta
hemoglobin
heterodimer
b
8-3
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
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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 (nonhydrolyzable ATP analogue)
 mitochondrial chaperonin (Hsp60) is single-ring; GroES from chloroplasts consists of
a fused dimer
8-5
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
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Group I chaperonin:
functional 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 --> 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
8-7
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.
8-8
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.
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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
GroEL function: in vitro
8-10
 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:
> 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)
> ability of GroEL/GroES to fold substrate
followed similar pattern (not shown)
Group II chaperonin system
eukaryal
 Group II chaperonins from the eukaryotic cytosol and archaeal cytosol are more
closely related to each other than they are to Group I chaperonins
 eukaryotic cytosolic chaperonin is called CCT or TRiC, for “Chaperonin containing
TCP-1” or “TCP-1 Ring Complex”. TCP-1 was the first subunit of CCT to be
characterized. It was found to be present within a hetero-oligomeric complex that
contained 8 different (related) chaperonin subunits
 8-fold symmetry (different than GroEL’s 7-fold)
 duplication of chaperonin subunits occurred early during evolution (2 billion years
ago), as all eukaryotes contain the same 8 orthologues
 involved in actin and tubulin biogenesis BUT folds a number of other proteins, e.g.,
VHL tumour suppressor, myosin, cyclin E, viral capsid, etc. and binds up to 10% of all
cellular proteins
archaeal
 the archaeal chaperonin, termed “thermosome”, consists of 1-3 different subunits,
depending on the archaeal lineage
 8- or 9-fold symmetry
 function in protein folding; during cellular stresses (>70% cellular protein!)
8-11a
8-11b
Group II chaperonin structure
side
view
of top ring
alpha-helical
protrusion
GroES
apical
domain
side
view
of bottom
ring
apical
domain
intermediate
domain
intermediate
domain
equatorial
domain
thermosome side view
GroEL
8 subunits
per ring;
4 alpha,
4 beta
subunits
thermosome top view
equatorial
domain
thermosome
comparison of GroEL/ES complex (one
subunit of GroEL, one subunit of GroES)
with single thermosome (alpha) subunit
8-11c
8-12
Group II chaperonin:
functional 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 ADP*Pi 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
MODEL WHICH INCORPORATES THE SUBSTRATE
Douglas et al. Cell 2011
8-13
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
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
8-14
8-15
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
8-16
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
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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)
8-18
Wheat small HSP
End view
Side view
Dodecameric structure
van Montfort et al. Nature Structural Biology (2001)
8-19
Hsp33: the redox chaperone
 exclusively bacterial; induced during oxidizing (stress) conditions in the cell
Hsp33
Hsp33
oxidizing conditions (e.g., H2O2)
Hsp33/Hsp33 dimer
 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.
8-20
Hsp33 substrate binding site
 two possible binding sites that are only available upon dimerization
 residues shown are highly conserved across bacterial Hsp33 proteins
 ‘multivalent’ binding—again?