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Journal of Amino Acids
Volume 2011, Article ID 843206, 16 pages
FunctionalSubunitsof EukaryoticChaperoninCCT/TRiC in
M. AnaulKabir,1WasimUddin,1Aswathy Narayanan,1PraveenKumarReddy,1
M. AmanJairajpuri,2Fred Sherman,3andZulfiqar Ahmad4
1Molecular Genetics Laboratory, School of Biotechnology, National Institute of Technology Calicut, Kerala 673601, India
2Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India
3Department of Biochemistry and Biophysics, University of Rochester Medical Center, NY 14642, USA
4Department of Biology, Alabama A&M University, Normal, AL 35762, USA
Correspondence should be addressed to M. Anaul Kabir, firstname.lastname@example.org
Received 15 February 2011; Accepted 5 April 2011
Academic Editor: Shandar Ahmad
Copyright © 2011 M. Anaul Kabir et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
Molecular chaperones are a class of proteins responsible for proper folding of a large number of polypeptides in both prokaryotic
and eukaryotic cells. Newly synthesized polypeptides are prone to nonspecific interactions, and many of them make toxic
aggregates in absence of chaperones. The eukaryotic chaperonin CCT is a large, multisubunit, cylindrical structure having two
identical rings stacked back to back. Each ring is composed of eight different but similar subunits and each subunit has three
distinct domains. CCT assists folding of actin, tubulin, and numerous other cellular proteins in an ATP-dependent manner. The
catalytic cooperativity of ATP binding/hydrolysis in CCT occurs in a sequential manner different from concerted cooperativity as
the central cavity. The CCT complex recognizes its substrates through diverse mechanisms involving hydrophobic or electrostatic
interactions. Upstream factors like Hsp70 and Hsp90 also work in a concerted manner to transfer the substrate to CCT. Moreover,
prefoldin, phosducin-like proteins, and Bag3 protein interact with CCT and modulate its function for the fine-tuning of protein
folding process. Any misregulation of protein folding process leads to the formation of misfolded proteins or toxic aggregates
which are linked to multiple pathological disorders.
The primary amino acid sequence of a protein contains all
the information necessary for protein folding and its biolog-
ical activity . However, in a normal cellular condition, a
nascent polypeptide chain faces a crowded environment and
there is a good possibility that protein will be misfolded and
will form aggregates that make the protein inactive, and in
certain cases it becomes toxic for the cell. Both the prokary-
otic and eukaryotic cells possess a family of proteins respon-
sible for binding to nascent polypeptide chains and help
them fold into biologically functional three-dimensional
structures, they are known as molecular chaperones, and
they vary in size and complexity [2–6]. Many of the molecu-
lar chaperones are induced in response to stress or heat, and
so they got the name Hsp (heat shock protein). Molecular
chaperones like Hsp90, Hsp70, Hsp40, and Hsp104 bind to
nascent polypeptide chain at hydrophobic regions which are
in a completely folded protein [7–10]. Molecular chaperones
have developed multiple and diverse tertiary and quaternary
structures to bind nonnative protein substrates. Though,
there is a lack of sequence similarity among different families
of chaperones and only a few of them are represented in
all three domains of life (bacteria, archaea, and eukaryote),
generally, they use convergent strategies to bind the sub-
strates. Crystallographic and other evidence show that many
chaperones including prefoldin, trigger factor, hsp40, and
hsp90 have clamp-like structures, possibly responsible for
the binding of nonnative substrates . Another class of
2Journal of Amino Acids
cylindrical-shaped chaperones, known as chaperonins, is
found to be conserved in all three domains of life and
assist the folding of many cytosolic proteins [12, 13]. In
some cases, the transient binding of nascent polypeptide
chain is sufficient for protecting its hydrophobic regions and
promoting its proper folding. However, for the folding of a
multidomain protein, more than one class of chaperones
might be involved, and they work in a concerted manner
to generate a protective passage. For example, nascent poly-
peptide chain coming out from ribosome will first bind to
Hsp70/Hsp90 which will help attain a quasinative structure
and then will be transferred to chaperonin CCT for its final
folding [14, 15]. Here we review the current status of under-
standing of protein folding by the chaperonin CCT complex
The chaperonins are large, multimeric, cylindrical pro-
tein complexes consisting of two stacked rings and each
ring has 7–9 subunits [2, 4, 16–18]. On the basis of amino
acid sequence homology, chaperonins have been categorized
into two groups, group I and group II [19–24]. Group
I is found in all eubacteria and endosymbiotic organelles
like mitochondria, chloroplasts, and related organelles like
hydrogenosomes and mitosomes whereas group II chap-
eronins are present in archaebacteria and in the cytosol
of all eukaryotes [2, 17, 25–27]. Here we will give brief
introduction to group I chaperonin and then will discuss
group II chaperonin, CCT.
The function of this group has been well studied using
GroEL/GroES from Escherichia coli. The genes for GroEL
and GroES were discovered in a mutagenic screen for genes
required for the growth of bacteriophage lambda and later
ditions [28–30]. GroEL/GroES has been the subject of exten-
sive structural and functional analysis for understanding
protein folding in vitro and in vivo [31–33]. GroEL is about
800kDa homooligomeric protein complex with ATPase
activity and is composed of two seven-membered rings of
57kDa subunits. Each ring has a central cavity aligned
with hydrophobic surfaces for the binding of unfolded or
denatured proteins [34–37]. The cochaperonin GroES is
a heptameric ring complex composed of 10kDa subunits
and caps the GroEL folding chamber [38–40]. The folding
cage generated by GroEL/GroES plays dual functions in
aggregation. Second, the folding process in the chaperonin
would be much faster than that of in a free solution . X-
ray studies of GroEL have revealed three distinct domains:
equatorial domain, apical domain, and intermediate domain
. The equatorial domain is responsible for most of the
intra- and intersubunit interactions as well as for the binding
of ATP and its hydrolysis. The apical domain encompasses
the entrance of central cavity and holds all the hydrophobic
residues required for substrate binding. The intermediate
domain connects both the domains and acts as a hinge for
the movements of apical domain which is induced upon
binding of ATP and its hydrolysis [43–45]. The substrate
binding residues present in the apical domain is also respon-
sible for binding of co-chaperonin, GroES, which is essential
for GroEL-mediated protein folding. The mechanism of
using different approaches [16, 24, 46, 47]. Briefly, protein
proteins and ATP at one end (the cis end) of GroEL, followed
by the binding of GroES to the same end. The binding
of GroES caps the entrance and releases the substrate to
the central cavity. The binding of GroES also promotes
ATP hydrolysis and the protein substrate gets folded in the
“Anfinsen cage” of the GroEL-GroES-ADP complex. The
binding of protein substrate and ATP to trans ring causes the
release of GroES and substrate .
In the group II chaperonins, both archaeal thermosome and
eukaryotic chaperonin containing TCP-1 (CCT; also known
as TCP-1 ring complex, TRiC) are being studied using
different techniques [49–52]. However, here we will confine
our discussion to CCT complex only. The subunit TCP-1
of CCT complex was first isolated from murine testes and
subsequently it was found to be constitutively expressed
in other mammalian cells, insects, and yeasts [53–57]. The
compelling evidence for CCT complex as a chaperonin came
from the observations of its involvement in the assembly
of actin and tubulin filaments [58–61]. Though, initially, it
was discovered as a folding machine for actin and tubulin,
later, it was found to be involved in the folding of 5–
10% of newly synthesized cytosolic protein substrates [12,
52, 62, 63]. Like GroEL system, CCT also binds ATP and
hydrolyses it during protein folding cycle, the CCT does not
have detachable GroES-like cochaperonin, rather, a flexible
protrusions located in the apical domain in each CCT
subunit acts as a lid and is responsible for closing the central
cavity [17, 64, 65].
CCT is a large, cylindrical, multimeric complex having cen-
tral cavity for binding unfolded or denatured polypeptides.
The structure of CCT is much more complex compared
to GroEL because each ring of CCT is composed of eight
[58–60]. Moreover, it has been observed that all the eight
subunits of CCT are essential in yeast . Now the question
is when homooligomeric GroEL is sufficient to fold an array
of substrates, why did CCT complex evolve eight different
subunits? Perhaps, one possible explanation could be that
certain specific combinations of subunits interact with
specific structural features or motifs of the protein substrates
and hence eight different subunits give large number of
combinations to accommodate a broad variety of substrates
for their folding [66, 67]. Furthermore, the presence of
eight different subunits encoded by eight paralogous genes
indicates that every subunit might have some specialized role
Journal of Amino Acids3
Figure 1: The intra-ring subunit arrangement of CCT. Both Greek
alphabets and Arabic numbering system have been used to denote
each of the subunits (this was made on the basis of ).
in the overall functioning of CCT. Phylogenetic analysis of
these eight subunits suggested that functional specialization
of the individual subunits took place in the early phase
of eukaryotic evolution and associated with its cellular
functions and became essential for its survival [68, 69].
The electron microscopy and single particle analyses show
that the domain structures of CCT are similar to those
of GroEL and archaeal thermosome, and three domains
(equatorial, apical, and intermediate domains) are present
in a single subunit . The mammalian subunits of CCT
are designated as CCTα, CCTβ, CCTγ, CCTδ, CCTε, CCTζ,
CCTη, and CCTθ which are corresponding to Cct1p-Cct8p
in yeast. Using biochemical and genetic approaches, it has
been shown that CCT is a single heterooligomer of eight
subunits and the arrangement of the subunits has a unique
pattern as shown in Figure 1 [70, 71]. The subunits in a ring
are arranged as Cct1p→Cct5p→Cct6p→Cct2p→Cct3p→
Cct8p→Cct4p→Cct7p in a clockwise orientation. The ar-
rangement of subunits in CCT ring also addressed by mod-
eling using existing data, and four possible models have been
proposed with clockwise and anticlockwise orientations;
however, the relative positions of the subunits in a ring re-
main same [71–74]. The phasing between two rings of CCT
has been addressed by three-dimensional reconstructions
generated by electron microscopy and using monoclonal
antibodies against Cct4p and Cct5p. It has been shown that
interring communications take place through two different
subunits in all the eight positions . Moreover, the
subunits associated with initiation and completion of the
folding cycle cluster together in both inter- and intraring
ed new positions in a ring for three subunits-Cct1p, Cct5p,
in a clockwise orientation . This observation identifies
the existence of two-fold axis between two rings. It reveals a
unique pattern of interring arrangement that generates three
heterotypic interring contacts (Cct3p-Cct4p, Cct2p-Cct5p
and Cct6p-Cct7p) and two homotypic interring contacts
(Cct1p-Cct1p and Cct8p-Cct8p). As the relative positions
of other five subunits remain same in both the models and
only three are having deviation in each of these models, fine-
tuning of biochemical and EM analyses will be required to
fix their positions. Recently, crystal structure analysis has
been done for CCT purified from bovine testis along with
its natural substrate, tubulin in the open conformation .
This structure showed that substrate interacts with CCT in
the loops of apical and equatorial domains. The organi-
zation of ATP binding sites indicates that the substrate is
(equatorial, intermediate and apical) of the eight subunits
of CCT found to be similar to those of α- and β-subunit
of thermosome. The thermosome equatorial domains can
be superimposed on their CCT homologues, though small
rearrangements in the orientation of helices and loops along
the equatorial domain were observed. In contrast to the
crystal structures of GroEL and thermosome, apical domains
of CCT showed a wide range of conformations, both in
the central cavity and in the aperture of the lid domains
[42, 78, 79]. This supports the existence of sequential,
hierarchical mechanism of conformational changes induced
3.2. Nucleotide Binding to CCT and Protein Folding. The
binding of ATP to the subunits of CCT and its hydrolysis is
absolutely required for the CCT-mediated folding of newly
synthesized polypeptide or misfolded protein substrates [58,
60, 80]. In the absence of ATP or in presence of ADP,
CCT remains in an open conformation in which substrate
binding sites in the apical domains are exposed and bind
the substrates. Addition of ATP induces the conformational
in lid structure to confine the protein substrate inside the
cavity and provides a secluded environment for its folding.
However, just addition of ATP to CCT does not promote
the lid closure asshown using non-hydrolyzableATP analogs
[65, 81]. Using non-hydrolyzable ATP analog (AMP-PNP), it
has been demonstrated that AMP-PNP-CCT binds to actin
and tubulin on one CCT ring [66, 82]. On the other hand,
when CCT is blocked in its ADP-Pistate, no CCT-protein
target complex was formed. This suggests that binding sites
in the apical domain of CCT will be available for target
proteins in ATP-CCT state but not in ADP-Pi-CCT state.
Therefore, nucleotide exchange and hydrolysis might be
working as a regulatory switch for the binding of target
proteins to CCT .
3.3. Lid Structure of CCT. The bacterial chaperonin GroEL
central cavity essential for protein folding . However, the
eukaryotic cytosolic chaperonin CCT/TRiC does not have
any homologue of GroES to cap the central cavity [17, 78].
The crystal structure of archaeal chaperonin thermosome
has shown the presence of protrusions emerging from the
apical domain and arranged in an iris-like β sheet which
is responsible for closing the central cavity . These
apical protrusions are unique to group II chaperonins, like
4Journal of Amino Acids
thermosome and CCT, and they have been proposed to
have GroES-like activity and act as a built-in lid that might
open and close in an ATP-dependent manner [17, 85].
Compelling evidence for the requirement of lid responsible
for encapsulating the unfolded polypeptides comes from the
observations that lidless chaperonins lose the ability to fold
stringent substrates [65, 86, 87]. The question is how does
this “built-in lid” functions for the closing and opening
of the central cavity for substrate binding and releasing?
Though there are striking similarities between group I and
group II chaperonins, the lid closure mechanism seems to
be quite different from each other. The binding of GroES
to GroEL occurs upon ATP binding to equatorial domain of
the GroEL subunits whereas the lid formation in eukaryotic
and archaeal chaperonins is triggered by the transition state
of ATP hydrolysis, suggesting nucleotide cycle dependent
mechanistic difference of lid closure . In one study, using
the Thermococcus chaperonin, it has been suggested that
ATP binding/hydrolysis causes independent conformational
changes in the subunits. However, complete closure of the
lid is induced and stabilized by the interactions of the helical
protrusions of different subunits .
3.4. Mutations Affecting ATP Hydrolysis. The high-resolution
crystal structure of GroEL in ATP-bound and -unbound
formed and that of GroES has identified an ATP binding
domain encompassing N- and C-terminus of GroEL sub-
units [42, 90, 91]. This domain contains highly conserved
GDGTT (residues 86–90) ATP binding residues along with
loop structural motifs, LGPKG (residues 31–35), ITKDG
(residues 49–53), and GGG (residues 414–416) which are
ogy analysis of CCT subunits with that of GroEL identified
almost identical ATP binding motif containing residues
GDGTT and other loop structural motifs suggesting their
conserved role in ATP binding/hydrolysis in prokaryotes as
well in eukaryotes. Though the conserved loop elements
appear to have certain common functions in ATP bind-
ing/hydrolysis, they could possess some functions which
are specific to certain loop elements in a subunit. This is
inferred from the observations that certain alleles of the
same gene, affecting residues from different conserved loop
structures have different degrees of cytoskeletal dysfunctions
[92, 93]. Besides, there might be functional hierarchy among
the paralogous motifs of different subunits. For example,
homologous replacement of the fifth conserved glycine
residue to glutamic acid in the LGPKG motif causes a lethal
phenotype in Cct2p whereas the same mutation in Cct1p
makes it heat sensitive in yeast Saccharomyces cerevisiae.
effect (Table 1 and Figure 2) [92, 94]. Furthermore, it has
GDGTT→AAAAA replacement in the Cct6p in S. cerevisiae
for Cct1p . From the above-mentioned experimental
data, it can be suggested that different subunits play different
role for ATP binding/hydrolysis in the CCT. This might
Table 1: Mutations in the conserved ATP binding/hydrolysis do-
Subunit Amino acid replacement
CS: Cold sensitive; HS: heat sensitive; TBZS: Thiabendazole sensitive;
TBZSS: Thiabendazole-hyper sensitive; NaClS: Sodium chloride sensitive.
be required for folding different substrates and modulating
intraring and interring interactions.
3.5. Substrate Recognition by CCT. One of the most well-
studied chaperonins is the GroEL from E. coli, and the
recognition of substrate by this chaperonin has been studied
to a large extent, and so we will briefly discuss substrate
recognition mechanism of GroEL before we go into the
details of substrate recognition by CCT complex. A number
of techniques have been used to unveil the mechanism of
substrate binding by GroEL. The localization of substrate
binding region appears to be at the entrance of central cavity
of GroEL which has been shown by electron microscopy
[34, 97]. The X-ray crystal structure revealed this substrate
binding region in each subunit and has been termed “apical
domain” . A systematic mutational study was used to
understand the role of different amino acids in this region,
and it revealed that A152, Y199, S201, Y203, F204, L234,
L237, L259, and V263 play important roles in binding the
substrates as the mutations in these residues in the apical
domain affect the substrates binding to GroEL severely .
Interestingly, most of these residues have hydrophobic side
chains which can generate a hydrophobic surface for the
binding of the substrate. On the other hand, mutations
of charged amino acids in the apical domain appear to
have no effect on the binding of the substrates. This
suggests that hydrophobic residues in the apical domain are
mainly responsible for creating hydrophobic surfaces in the
central cavity for binding of substrates through hydrophobic
interactions. However,thesingle-residue replacementsin the
intermediate domain (I150E, S151V, A152E, A383E, A405E,
and A406E) exert global effect on the functioning of GroEL
. All of these mutants showed severe defect in ATPase
Journal of Amino Acids5
31354953 8690414 416
41 5992 416
40 5891 413
Figure 2: Comparison of highly conserved ATP binding/hydrolysis motifs in equatorial domain. GroEL, chaperonin of E. coli; Cct1p-Cct8p
subunits of CCT complex of yeast S. cerevisiae; Cctα-hu, Cct1α subunit from human and Thr-β, β subunit from Acidianus tengchongensis.
Starting from E. coli (homooligomeric chaperonin) to human (heterooligomeric chaperonin), all the chaperonin subunits have maintained
conserved regions and any changes would have severe effects.
activity though they fall outside the ATP-binding domain.
Also the mutants at positions, 150, 151, 383, and 405 could
bind polypeptide but the release of the polypeptide was
severely affected. On the other hand, D87K/D87N mutation
in the conserved domain, GDGTT, in the equatorial domain,
lost the ATPase activity completely, though it has the ability
to bind ATP. It has also reduced the ability to bind polypep-
tide; however, there was a complete block of polypeptide
Several techniques have been used to implicate the im-
and the substrates [98–102]. However, there are some
exceptions to this substrate recognition principle and certain
other forces such as electrostatic interactions might play an
important role as well for binding the substrate to GroEL
Although group I chaperonin (GroEL) and group II
chaperonin (CCT) have double ring structure and share se-
quence similarities, they differ from each other in two major
aspects. First, group I chaperonin is composed of identical
subunits and has seven subunits per ring whereas group II
chaperonin is composed of 2–8 paralogous subunits with
30–40% homology to one another and each ring has 8–9
subunits [16, 17, 26, 106, 107]. For example, CCT is com-
posed of eight paralogous subunits [52, 70]. However,
the functional relevance of this subunit diversity is not
well understood. As the sequence divergence in the apical
domain is more among the paralogous subunits, it has been
hypothesized that different subunit in CCT has different
do not have GroES-like cofactor; however, it possesses a
helical protrusion that acts as “built-in lid.” These two major
eukaryotic proteins. The structural and mechanistic differ-
ences between two groups might have profound functional
impact on the substrate specificity [13, 109]. For example,
several eukaryotic protein including actin and tubulin can
be folded by CCT only whereas the bacterial proteins which
require the assistance of GroEL for their folding, are not able
to fold in eukaryotic cytosol [13, 58, 109, 110].
The specificity of GroEL and CCT towards the substrates
is thought to be due to chemical nature of their interactions
with substrates. It has been well established that GroEL
recognizes the exposed hydrophobic surfaces of unfolded
substrates [111–113]. On the other hand, CCT subunits
6Journal of Amino Acids
possess specific binding sites for unique polar motifs of
certain cellular proteins [82, 114–116]. However, using a
biochemical approach, it has been shown that for the
binding of actin, von Hippel-Lindau tumor suppressor and
Gβ WD-40 protein to CCT, hydrophobic interactions are
involved [117–119]. In the absence of well-defined structural
surfaces or motifs present in the substrates of CCT, three
model proteins, actin, tubulin, and von Hippel-Lindau
tumor suppressor have been studied thoroughly to find the
recognition sites present in these substrates as well as in the
interacting subunits of CCT.
3.5.1. Recognition Sites in Actin and Tubulin. Several studies
have pointed out that the nature of actin and tubulin
conformations bound to CCT are not of any nonspecific
structures as in the case of GroEL substrates rather they
must have some kind of defined, quasinative conformations
before they are recognized by CCT for the final steps of their
folding [80, 120–122]. The quasinative conformation may be
achieved themselves or they may be guided by prefoldin kind
of cochaperonin to reach that conformation. The atomic
structure of actin has shown the presence of two domains,
small and large . The three-dimensional reconstruction
analysis of alpha actin with CCT using electron microscopy
has shown that CCT interacts with these two domains of
actin by two specific and distinct interactions. The small
domain of actin interacts with Cct4p subunit whereas
large domain interacts with either Cct2p or Cct5p (both
are 1,4 position with respect to Cct4p). This observation
led to the suggestion that CCT interacts with actin in
subunit-specific and geometry dependent-manner .
The three-dimensional reconstruction analysis combined
with immunomicroscopy and screening study using actin
peptide arrays have identified residues in four regions of
actin molecule [114–117, 124]. Two of these regions ( R37-
D51 and R62-T66) are located at the tip of small domain
and interacts with Cct4p subunit of CCT. The other two
regions (E195-R206 and T229-I250) are present at the tip
of large domain and interact with Cct2p or Cct5p subunit.
Mutational analysis coupled with electron microscopy and
biochemical assay have shown that major determinants of
actin binding to CCT are present at the tip of the large
domain [116, 125]. A mutation (G150P) in the conserved
putative hinge region between small and large domains
Furthermore, electroncmicroscopic studies have shown that
actin interacts with Cct2p or Cct5p subunits rather than
Cct4p subunit. It was thought that Cct2p and Cct5p might
have highest substrate affinity, and this possibility has been
strengthened by immunoprecipitation experiments of actin-
CCT complexes .
On the other hand, the interaction of tubulin with CCT
seems to be much more complex and it does not confine
to a few regions of tubulin rather it is spread along its
entire sequence and interacts with several domains at a time
[82, 117, 122, 124, 126, 127]. Several studies have shown that
CCT binding sites in tubulin are present in loops exposed to
the surface of native protein [67, 82, 128, 129]. Of the eight
binding sites present in tubulin, three are located at N-
terminal domain and five are placed in C-terminal domain.
The N-terminal binding sites are T33-A57, S126-Q133, and
E160-R164. Immunomicroscopic experiments have shown
that residues T33-A57 interact with Cct1p or Cct4p whereas
the residues S126-Q133 and E160-R164 interact with Cct7p
or Cct8p . The interaction of C-terminal domain of
tubulin with CCT is much more complex than that of N-
terminal domain. Five putative segments present in the C-
terminal domain responsible for interaction with CCT are
The segments T239-254 and P261-H266 interact with Cct6p
or Cct3p subunits, the segments S277-V288 and V355-P359
interact with Cct5p or Cct2p whereas the segment W407-
E417 interacts with Cct2p or Cct8p subunits .
Though it was suggested that tubulin binding sites could
thought to be hot spot for the binding of tubulin to CCT
and it might have higher affinity to CCT compared to other
sites [117, 122, 126, 127, 130]. Recently, Jayasinghe et al.
used a computational approach to pinpoint the interactions
between gamma subunit of CCT and its stringent substrate
beta-tubulin. It has been shown that the substrate binding
sites in CCT are composed of helical region (HL) and helical
protrusion region (HP). Interaction of substrate at helical
region involves both hydrophobic and electrostatic contacts
while binding to helical protrusion is stabilized by salt bridge
3.5.2. Recognition Sites in von Hippel-Lindau (VHL). The
tumor suppressor protein von Hippel-Lindau (VHL) has
been extensively studied from substratepoint of view of CCT
and has been shown to be an obligate substrate of CCT
[14, 132]. VHL is a subunit of a ubiquitin ligase complex
that targets cellular proteins, like HIF-1α, for proteolysis
[133, 134]. Loss of function mutations in VHL is responsible
for the tumor formation in kidney, adrenal glands, and
central nervous system [135, 136]. VHL is composed of
213 amino acid residues of which 55-amino acid domain
(100–155 residues) is necessary and sufficient for binding to
CCT [132, 137]. Interestingly, most of the mutations in this
domain are responsible for VHL diseases [138–140]. Using
alanine-scanning mutagenesis procedure, the 55-amino acid
segment has been completely analyzed to identify minimal
regions responsible for CCT binding. This has revealed that
two small regions of VHL, amino acid 116–119 (Box1) and
148–155 (Box2; Figure 3) are absolutely required for stable
binding with CCT .
Furthermore, contribution of individual amino acid in
these two boxes has been evaluated using single alanine
substitution mutants. This analysis has shown that alanine
replacement of W117 and L118 within Box1 or F148, I151,
L153, or V155 within Box2 (Figure 3) substantially reduce
the binding of VHL to CCT . Though these two boxes
are distant in the primary structure, they are located in the
adjacent strands within the β sheet domain of folded VHL
and the side chains of two boxes are projected in the same
Journal of Amino Acids7
1 100 116119 148155213
Figure 3: von Hippel-Lindau (VHL) protein containing two boxes,
Box1 and Box2, required for binding to CCT. VHL is 213 amino
acids long protein. Amino acid residues 100–155 constitute TRiC
binding domain (TBD; this diagram was made on the basis of
direction to generate a hydrophobic surfaces required for its
interaction with CCT.
3.6. Allosteric Regulations in CCT. Allosteric regulation plays
an important role for transitions between different func-
tional states among the molecular machines in response
to changes in environmental conditions. As it was strongly
believed that allosteric regulation of chaperonins is crucial
for assisting the protein folding, the chaperonins GroEL and
CCT complex were studied from allosteric point of view
to understand their functioning [47, 141]. The allosteric
transitions of GroEL can be described by a nested allosteric
model in which each of its rings is in equilibrium between a
T state and an R state. The T state has low affinity for ATP
and high affinity for unfolded substrate proteins whereas
R state possesses high affinity for ATP and low affinity for
unfolded substrates [142–144]. It has been shown that T and
R states interconvert in a concerted manner in accordance
with Monod-Wyman-Changeux (MWC) model of coopera-
tivity . However, in the presence of high concentration
of ATP, GroEL ring switches from TT state to RR state
via TR state in a sequential manner in accordance with
Koshland-N´ emethy-Filmer (KNF) model of cooperativity
. Though the overall structure of both GroEL and CCT
is similar, CCT is different from GroEL with respect to its
subunit composition and so, it was important to unveil
CCT. Both biochemical and genetic approaches have been
adopted to understand this mechanism. Kinetic studies have
shown that CCT undergoes two ATP-dependent transitions
ric particle in which one ring will have slight conformational
changes whereas the other ring undergoes a substantial
movement in the apical and equatorial domains . Using
the powerful yeast genetics, it has been suggested that intra-
it occurs in a sequential manner around the ring. This was
inferred from the suppression analysis of different mutant
alleles of Cct1p, Cct2p, Cct3p, and Cct6p . Moreover,
EM analysis has shown two important differences between
GroEL and CCT. First, a lot of conformational heterogeneity
has been observed in the apo state of CCT but not in GroEL.
Second, ATP-induced conformational changes take place in
a sequential manner in CCT whereas concerted mechanism
is observed for GroEL . Biochemical as well as genetic
analysis data suggested that ATP-induced conformational
changes in CCT take place in the order Cct1p→Cct3p→
Cct2p→Cct6p [70, 150].
3.7. Cochaperones of CCT. The role of CCT has been well
established for the folding of a large number of proteins.
However, it was not clear in the beginning whether CCT
alone is sufficient for the folding of nascent chains to their
maturity or other components are also required. Later, it
was found in a genetic screen during the identification
of synthetic lethals for gamma-tubulin that GimC, also
known as prefoldin (PFD), participates in the maturation of
cytoskeletal proteins . Using biochemical approach, it
has been shown that prefoldin plays an important role for
the formation of functional actin and tubulin by transferring
unfolded protein substrates to CCT . The role of
prefoldin (GimC) has been established in the folding of
actin using chaperone trap and suggested that prefoldin acts
along with CCT for the maturation of the substrate protein
. Using in vitro transcription/translation of actin, it has
been shown that unfolded actin polypeptide chain remains
bound to prefoldin until it is transferred to CCT. Similar
observations were made for the maturation of α- and β-
tubulin as well . Prefoldin is a heterohexameric protein
complex that exits both in archaea and eukaryotes. However,
eukaryotic prefoldin is composed of six different subunits
whereas archaeal prefoldin has only two different kinds of
subunits: α and β subunits. It is possible that like CCT,
eukaryotic prefoldin has been developed to more complex
structure from simpler archaeal form to heterohexameric
structure to participate in the more complex protein folding
processes. Three-dimensional reconstruction of CCT with
prefoldin based on electron microscopy analysis has shown
that prefoldin interacts with each of CCT rings in a unique
conformation with two specific subunits that are placed
in a 1,4 arrangement. Therefore, it is highly desirable that
Cct2 subunits or Cct4 and Cct5 subunits . A large body
of evidence show that heterohexameric complex of prefoldin
uses its jellyfish or octopus-like structure to grip nonnative
protein substrates and transfer it to CCT for proper folding
[74, 155, 156].
Another set of proteins implicated in the regulation of
CCT function are phosducin-like proteins (PhLPs) which
were originally identified as modulators of heterotrimeric G
protein signaling . Subsequently, they were found to
play an important role in the regulation of CCT function
[158–162]. PhLPs are subdivided into three families like
PhLP1, PhLP2, and PhLP3 and they share structural simi-
larities at N-terminal helical domain, a central thioredoxin-
like fold, and a C-terminal extension . PhLP1 has been
shown to have inhibitory effect on CCT and this may be
required for regulating the protein folding capacity of CCT
. Electron microscopy reconstruction of mammalian
CCT: PhLP1 has demonstrated that PhLP1 binds to apical
8Journal of Amino Acids
domains of several chaperonin subunits . Furthermore,
the interaction of PhLP2 with CCT was suggested in
proteome-wide studies. To substantiate this observation, in
vitro study was done using human PhLP2A and has been
shown that it does inhibit the folding of actin and forms
ternary complex with CCT and actin in mammalian system
. However, recent study has suggested stimulatory role
of PLP2 in yeast S. cerevisiae . It has been shown that
PLP2-CCT-ACT1 complexes produce 30-fold more actin
than CCT-ACT1 complexes in a single ATP driven cycle.
PLP2 itself can bind to actin through its C-terminal of
thioredoxin fold and CCT-binding subdomain 4 of actin
. The inhibitory effect of human PDCL3, an orthologue
extension of that of PLP2 of yeast . Therefore, it
seems that higher eukaryotes have developed another level of
regulatory control of CCT by phosducin-like proteins. The
third member of this family, PhLP3, has been shown to bind
CCT as well. The PhLP3 forms ternary complex with CCT
and actin or tubulin, and does inhibit the folding process.
It has been suggested that this negative impact is not due to
direct competition for substrates rather by diminishing the
ATPase activity of CCT by PhLP3 . Moreover, in vivo
experiments have shown that yeast PhLP3 might coordinate
the proper biogenesis of actin and tubulin with prefoldin
normal function of G protein signaling [161, 162, 164, 166].
Therefore, both prefoldin and phosducin-like proteins are
working as co-chaperones to modulate the function of CCT.
In another study, it has been shown that caveolin-1
can interact with CCT and modulates its protein folding
activity . The caveolin-1-TCP interaction involves the
first 32 amino acids of the N-terminal segment of caveolin.
Phosphorylation at tyrosine residue 14 of caveolin-1 induces
the detachment of caveolin-1 from CCT and activates actin
folding . Recently, Bag3 protein has been identified as
another co-chaperone of CCT. Bag3 protein is known as co-
chaperone of Hsp70/Hsc70 and involved in the regulation
of various cell processes, such as apoptosis, autophagy, and
cell motility. Using RNAi, it has been shown that strains
lacking Bag3 activity slowed down-cell migration and also
influenced the availability of correctly folded monomeric
actin . Altogether, it shows that CCT is highly regulated
by cochaperones for its folding activity of actin and other
proteins and the interaction of different co-chaperones with
CCT decides the fate of the final folding process.
3.8. Phosphorylation of CCT. Recently, it has been demon-
strated that p90 ribosomal S6 kinase (RSK) and p70 ribo-
somal S6 kinase (S6K) can phosphorylate CCT in response
to tumor promoters or growth factors that activate the Ras-
mitogen activated protein kinase (MAPK) pathway .
RSK and S6K phosphorylate Ser-260 of Cct2p (Figure 4).
Furthermore, it has been shown that Cct2p plays an impor-
tant role in regulating cell proliferation and especially the
phosphorylation of Cct2p at Ser-260 contributes substan-
tially to this . Though Cct2p has been implicated in this
process, how the phosphorylation of Cct2p modulates the
function of CCT is not clear. However, there could be two
implications of this phosphorylation. First, phosphorylated
Cct2p subunit itself might be interacting with certain factors
of Ras-MAPK and PI3K-Mtor-pathways and regulate the
cell proliferation in response to multiple agonists in diverse
mammalian cells. Second, the phosphorylation of Cct2p
subunit of CCT might change the folding rate and reduce
the stress-related unfolded proteins in the cell. On the
other hand, it has been shown that a fraction of GroEL is
phosphorylated at least one phosphate at each of its subunits
enhances 50–100 -fold capacity of this chaperonin to bind
to denatured proteins. Possibly, the phosphorylated form of
GroEL might be responsible for refolding or degradation of
certain damaged polypeptides . In another study, it has
been shown that GroEL of Thiobacillus ferrooxidans is phos-
phorylated in response to phosphate starvation suggesting
its role in sensing and regulating stress responses in bacteria
. It is plausible that CCT carrying phosphorylated form
of Cct2p might be playing similar roles in eukaryotes.
3.9. Cooperation of CCT with Upstream Chaperones. Many
newly translated proteins may interact with several different
chaperones before they reach their biologically functional
three-dimensional structures. One of the most abundant
molecular chaperones is Hsp70 which was found to associate
with CCT in vivo suggesting their cooperation in protein
folding [59, 132]. In vitro experiments were performed
to elucidate the cooperative nature of Hsp70 and CCT
using mammalian cell-free lysates. It has been shown that
short chains of actin and firefly luciferase can interact with
Hsp70 whereas longer ones interact with CCT [173, 174].
Besides, the cooperation between prefoldin (GimC) and
CCT were found for folding of actin and tubulin [152–
154]. Using three-dimensional reconstruction of CCT:PFD
based on cryoelectron microscopy, it was shown that pre-
foldin binds to CCT through two specific subunits .
Moreover, several studies have also suggested the interaction
between phosducin-like proteins and CCT indicating their
cooperative nature for protein folding [158, 159, 165]. The
interaction of caveolin-1 with CCT also modulates the
protein folding function of CCT . Furthermore, it has
been shown that sequential cooperation between Hsp70 and
Hsp90 plays an important role for the folding of steroid
hormone receptors and kinases [175, 176].
From the above-mentioned experimental evidence, it
appears that cooperation between different chaperones is a
central principle to the protein folding process. However,
cooperation may not be required for each newly synthesized
chaperones are recruited to the protein synthesis machinery,
and as soon as the polypeptide chains are coming out, they
are protected by these chaperones including CCT. It has been
demonstrated that chaperones can bind to ribosome-bound
polypeptide chains in both prokaryotes and eukaryotes [173,
174, 177–183]. It seems that the cellular proteins follow
different chaperone-dependent and chaperone-independent
Journal of Amino Acids9
Helical protrusion of Cct2p
Figure 4: Schematic of Cct2p phosphorylation by p90 ribosomal S6 kinase (RSK) and p70 ribosomal S6 kinase (S6K) and the function of
CCT containing phosphorylated Cct2p.∗Indicates the S-260 position in the helical protrusion of Cct2p (this schematic is made based on
pathways for reaching their biologically functional three-
dimensional structures. The proteins in the chaperone-
independent pathway will probably be small proteins and
they can fold without any help from any chaperones. The
ferent strategies depending upon the nature of the proteins.
First, newly synthesized proteins will be bound by Hsp70,
Hsp90, or other small chaperones transiently at the exposed
hydrophobic regions present on the surface of nonnative
Second, some proteins will be bound by Hsp70 and Hsp90
sequentially and will proceed for folding. Third, a small
fraction of newly synthesized polypeptide chains will bind
sequentially to Hsp70, Hsp90, and prefoldin/phosducin-like
proteins and then transferred to CCT for final folding.
3.10. Protein Misfolding Diseases and CCT. The concerted
and cooperative action of a large number of molecular
biologically functional protein molecules. The failure in this
essential proteins and may cause a severe effect on the overall
functions in a cell. Now it has been well recognized that
protein aggregation and misfolding are the root causes for
many diseases known as “protein misfolding” or “protein
conformational” diseases . The toxic effect of the ag-
gregated or milfolded protein could be because of gain-
of-function which will have titrating effect on interacting
proteins or due to the loss of function of misfolded proteins
[185, 186]. Aggregation process is a multistep process having
stable or metastable intermediates that lead to the formation
proteins and result in the formation of inclusion bodies or
plaques that deposit outside or inside the cells [187–189].
A large body of evidence shows that the intermediates of
the aggregate formation process are responsible for disease
pathogenesis rather than the final products which may be
of diffuse polyglutamine- (polyQ-) expanded huntingtin is
thought to be cause of cell death in Huntington’s disease and
inclusion bodies could enhance the survival . Similarly,
proteinaceous deposits or inclusion bodies are found to be
not associated with toxicity in Parkinson’s and Alzheimer’s
Though chaperonins are generally responsible for
maintaining the cellular protein homeostasis, they are now
implicated in the pathogenesis of misfolding human diseases
as well. Both the group I and group II chaperonins are found
to be participating as modulators of misfolding diseases.
For example, inactivation of mitochondrial Hsp60 is
responsible for hereditary spastic paraplegia, a late-
onset neurodegenerative disease [195, 196]. Recent studies
have clearly shown that polyQ-expanded huntingtin is a
potential substrate of CCT [197–199]. Moreover, an RNA
interference screen in C. elgans has identified six out of
eight subunits of CCT as suppressors of polyQ aggregation,
suggesting that CCT can bind polyQ and inhibit the
formation of toxic aggregates . It has been shown that
10 Journal of Amino Acids
overexpression of Cct1p of CCT was effective at inhibiting
huntingtin aggregation and subsequently increased the
viability . On the other hand, the deletion of Cct6p of
CCT increases the huntingtin aggregation and toxicity [52,
199]. Generally, the CCT substrates are large, hydrophobic
proteins containing the regions with β-strand propensity,
[63, 117, 118, 201]. Aggregation of these proteins may begin
with conformational transition from native monomer to
mature amyloid fibrils [197, 202, 203]. Therefore, it is quite
possible that CCT binds directly to β-sheets and protects
the protein from being aggregated which can be otherwise
toxic for the cell. It has been shown that overexpression
of certain subunits of CCT can protect from misfolding
diseases. Therefore, drug-mediated induction of molecular
chaperones can be considered as one of the methods for
treating these diseases . Otherwise, certain CCT subunits
can be injected di-rectly on regular basis like insulin in case
of diabetic patients and misfolding diseases can be handled.
Molecular chaperones are crucial for the production of bio-
logically functional three-dimensional protein structures. A
large number of molecular chaperones are present in all
the three kingdoms of life implying their importance in
biological system. Chaperonins are cylindrical structures
having central cavity for encapsulating unfolded protein
substrates and assist in protein folding in an ATP-dependent
manner. The chaperonin CCT is composed of eight different
but related subunits of differential functional hierarchy
in which catalytic cooperativity of ATP binding/hydrolysis
takes place in a sequential manner rather than concerted
cooperativity as found in GroEL. Moreover, substrate recog-
nition in CCT takes place through diverse mechanisms
involving hydrophobic and electrostatic interactions. For
the fine-tuning of protein process, many cochaperones like
prefoldin, phosducin-like proteins act as upstream factors
and transferthe substrateto CCT. These upstreammolecular
chaperones and chaperonins might be responsible for gener-
ating a protective chaperone cage for the newly synthesized
polypeptide chains to minimize the chance of aggregation
and misfolding. Recently, it has been shown that certain
CCT subunits are phosphorylated in response to tumor
promoters or growth factors suggesting the possible roles
of different kinases and possible certain phosphatases in
regulating the activity of CCT. Any abnormal function posed
by any chaperone at any stage of protein folding might
have severe consequences. Many mutations in the molecular
chaperones are now linked to Parkinson’s and Alzheimer’s
diseases. Better understanding of chaperonin CCT and other
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