Post-nascent-chain binding chaperones- Chaperonins (bacterial GroEL, eukaryotic CCT, archaeal thermosome)- Small heat-shock proteins (Hsps)- Hsp33

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Chaperones involved in folding (II)Chaperones involved in folding (II)

A chaperone for -hemoglobinalpha-betahemoglobinheterodimer

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alpha-hemoglobin stabilizing protein (AHSP)

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 chaperonin systemGroEL/GroES chaperonin system

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

crystal structureof E. coliGroEL/GroES

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GroEL/GroES structureGroEL/GroES 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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GroEL subunit structureGroEL subunit structure

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

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Group I chaperonin:Group I chaperonin:functional cyclefunctional cycle

Paper presentation (next 3 slides):

Farr et al. (2000) Multivalent binding of nonnative substrate proteins by the chaperonin GroEL. Cell 100, 561-573.

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)

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GroEL mechanism of actionGroEL mechanism of action

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: GroEL function: single polypeptidesingle polypeptide

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

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GroEL function: GroEL function: in vivoin vivo

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)

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GroEL function: GroEL function: in vitroin vitro

8-11aGroup II chaperonin systemGroup II chaperonin system

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

eukaryal

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!)

archaeal

Group II chaperonin structureGroup II chaperonin structure8-11b

thermosome side view

thermosome top view

comparison of GroEL/ES complex (one subunit of GroEL, one subunit of GroES) with single thermosome (alpha) subunit

GroES

GroEL

apicaldomain

thermosome

sideviewof top ring

sideviewof bottomring

8 subunitsper ring;4 alpha,4 betasubunits

equatorialdomain

intermediatedomain

apicaldomain

intermediatedomain

equatorialdomain

alpha-helicalprotrusion

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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

ATPADP transition somehow causes large conformational change

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Group II chaperonin:Group II chaperonin:functional cyclefunctional cycle

Douglas et al. Cell 2011

MODEL WHICH INCORPORATES THE SUBSTRATE

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

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CCT-actin em reconstructionCCT-actin em reconstruction

FtsA, actin homologue FtsZ, tubulin homologue

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

Evolution of eukaryotes

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Evolution of chaperonins, prefoldin Evolution of chaperonins, prefoldin and actin/tubulinand actin/tubulin

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

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Small heat-shock proteinsSmall heat-shock proteins

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

- sizes of small Hspsrange from 150 kDato 800 kDa

- smallest functionalsmall Hsp is a nonamer(trimer of trimer)

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Small Hsp crystal structureSmall Hsp crystal structure

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)

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Small Hsp surface viewSmall Hsp surface view

Wheat small HSPWheat small HSP

van Montfort et al. Nature Structural Biology (2001)

End view Side view

Dodecameric structure

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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

Hsp33Hsp33

Hsp33/Hsp33 dimeroxidizing conditions (e.g., H2O2)

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

Jakob et al. (1999) Cell 96, 341.

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Hsp33: the redox chaperoneHsp33: the redox chaperone

two possible binding sites that are only available upon dimerization

residues shown are highly conserved across bacterial Hsp33 proteins

‘multivalent’ binding—again?

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Hsp33 substrate binding siteHsp33 substrate binding site