GroEL-GroES system

The bacterial chaperonin GroEL and its cochaperone GroES belong to the Hsp60/Hsp10 (aka Cpn60/Cpn10)
family of heat shock proteins. Bacterial GroEL is an ≈ 800-kDa hollow cylinder consisting of two seven-subunit
rings stacked back to back. GroEL uses a set of hydrophobic residues located at either end of the cylinder to
capture non-native substrate and to interact with its co-chaperone GroES, a 70-kDa, dome-shaped
homoheptamer. In vivo, binding of client proteins occur on the ring of GroEL that is not complexed to GroES
(known as the trans ring) and involves multiple contacts with the substrate that lead to nanomolar dissociation
constants. GroEL can fold proteins up to 60-kDa in size and appears to exhibit a preference for compact
intermediates consisting of two or more domains with alpha/beta-folds that are enriched in hydrophobic and
basic residues.

GroEL-mediated protein folding is believed to involve the following sequence of events: (1) substrate binding
to the nucleotide-free trans ring; (2) binding of 7 ATP molecules and encapsulation by GroES which results in
substrate release in an enlarged and now hydrophilic cavity as well as ejection of bound ADP and GroES from
the opposite ring; (3) substrate folding timed by the hydrolysis of ATP (≈ 10s); and (4) release of GroES, ADP
and either properly folded protein or folding intermediate caused by the binding of 7 ATPs and fresh substrate
to the opposite ring. If the ejected substrate still exhibits significant surface hydrophobicity, it will be re-
captured by GroEL and the above cycle repeated until a correct conformation is reached.

Energy from the hydrolysis of the ATP allows for structural alterations to the chamber, changing it from
hydrophobic to hydrophilic. This change allows the protein to refold. After a short time, the protein is ejected,
either folded or unfolded , and the cycle repeats.

ClpB

ClpB belong to the Hsp100 family of ring-forming heat shock proteins whose role is to either degrade
unfolded/misfolded proteins, or to solubilize and reactivate aggregated proteins. ClpB, which functions
independently of a protease component, is a hexameric ATPase involved in protein disaggregation. This
activity is essential for cells to transiently survive extreme thermal stress. Current models hold that the DnaK-
DnaJ-GrpE system plays a critical role in the early stages of the disaggregation reaction by facilitating ClpB-
mediated extraction of single molecules from aggregates. The captured substrate is next threaded through the
1.6 nm central pore of ClpB where it undergoes net unfolding in a mechanical process that consumes ATP. As it
exits the ClpB channel, the unfolded polypeptide may spontaneously refold or be transferred to the DnaK-
DnaJ-GrpE and/or GroEL-GroES for chaperone-assisted refolding.

ClpB contains a highly mobile N-terminus domain that surrounds its pore and contributes to enhanced
disaggregation activity against certain substrates. The N domain is however dispensable for aggregate
solubilization (in fact E. coli synthesizes two versions of ClpB, one containing and one lacking an N domain,
from the same mRNA). On the other hand, the ClpB M domain, a coiled coil structure that projects laterally
from the top of the ring, appears to play an essential role in the disaggregation process by coupling DnaK-DnaJ-
GrpE aggregate "feeding" and ClpB threading motor activity.

Hsp33

Hsp33, which was identified on the basis of its thermal induction, is a redox-regulated chaperone that
stabilizes proteins unfolded by severe oxidative stress. Under balanced growth conditions, Hsp33 is a reduced
monomer that coordinates a zinc atom via four conserved cysteine residues. When cells are exposed to
reactive oxygen species – a situation that commonly accompanies heat shock – the cytoplasm becomes more
oxidizing and Hsp33 monomers form intramolecular disulfide bonds. Zinc release and conformational
rearrangement of the protein's C-terminus into an unstructured form lead to dimerization. The Hsp33 dimer
now exhibits chaperone activity and can capture unfolded proteins via its N-terminal substrate binding
domains. The thioredoxin and glutaredoxin systems rapidly reduce Hsp33 disulfides in a process that does not
cause substrate release but primes the chaperone for fast inactivation. Upon return to nonstress conditions,
DnaK-DnaJ engage the bound substrate and refold it alone or with the help of GroEL-GroES.