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181© Springer International Publishing AG 2018
A. A. A. Asea, P. Kaur (eds.), Regulation of Heat Shock Protein Responses, Heat
Shock Proteins 14, https://doi.org/10.1007/978-3-319-74715-6_8
Chapter 8
Molecular Chaperones: Structure-Function
Relationship andtheir Role inProtein Folding
BhaskarK.Chatterjee, SaritaPuri, AshimaSharma, AshutoshPastor,
andTapanK.Chaudhuri
Abstract During heat shock conditions a plethora of proteins are found to play a
role in maintaining cellular homeostasis. They play diverse roles from folding of
non-native proteins to the proteasomal degradation of harmful aggregates. A few
out of these heat shock proteins (Hsp) help in the folding of non-native substrate
proteins and are termed as molecular chaperones. Various structural and functional
adaptations make them work efciently under both normal and stress conditions.
These adaptations involve transitions to oligomeric structures, thermal stability,
efcient binding afnity for substrates and co-chaperones, elevated synthesis during
shock conditions, switching between ‘holding’ and ‘folding’ functions etc. Their
ability to function under various kinds of stress conditions like heat shock, cancers,
neurodegenerative diseases, and in burdened cells due to recombinant protein pro-
duction makes them therapeutically and industrially important biomolecules.
Keywords Chaperone assisted folding · Heat shock · Molecular chaperones ·
Protein folding · Structure-function of chaperones
Abbreviations
ACD α-crystallin domain
ADP Adenosine di-phosphate
ATP Adenosine tri-phosphate
CCT Chaperonin containing TCP-1
CIRCE Controlling inverted repeat of chaperone expression
B. K. Chatterjee · S. Puri · A. Sharma · A. Pastor · T. K. Chaudhuri (*)
Kusuma School of Biological Sciences, Indian Institute of Technology Delhi,
HauzKhas, New Delhi, India
e-mail: tkchaudhuri@bioschool.iitd.ac.in
Bhaskar K. Chatterjee, Sarita Puri, Ashima Sharma, and Ashutosh Pastor authors are equally
contributed.
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CNX Calnexin
CRT Calreticulin
CS Citrate synthase
ER Endoplasmic reticulum
ERAD Endoplasmic reticulum associated degradation
FRET Fluorescence energy resonance transfer
HOP HSP90/HSP70 organizing protein
HSC70 Heat shock cognate
HSEs Heat shock elements
HSFs Heat shock response and specic transcription factors
Hsp Heat shock proteins
HSP Heat shock protein family
HSR Heat shock response
MalZ Maltodextrin glucosidase
NAC Nascent chain associated complex
NEF Nucleotide-exchange factors
NTD n-terminal domain
PBD Peptide binding domain
PPIase Peptidyl-prolyl isomerases
PTP Permeability transition pore complex
RAC Ribosome associated complex
RuBisCO Ribulose-1,5-bisphosphate oxygenase-carboxylase
SHR Steroid hormone receptors
sHsp Small heat shock proteins
sHSP Small heat shock protein family
TF Trigger factor
TPR Tetratricopeptide
TRiC TCP-1 ring complex
UPR Unfolded protein response pathway
8.1 Introduction
Living systems respond to threatening conditions at multiple levels in their quest for
survival. It may in the form of a ght or ight response, which is a result of any
imminent physical threat either to an organism or their inner homeostasis. For
example the temperature, ionic and sugar balance are regulated within a xed range
in our bodies and are probably optimized by evolutionary mechanisms. Similarly,
homeostasis is alsomaintained at the cellular level and maintaining such a balance
is imperative for the survival and efcient functioning of the cell. One of the major
homeostasis mechanisms operating at the cellular level is the protein homeostasis,
commonly referred to as proteostasis (Balch etal. 2008). Starting with maintaining
the structural organization of a cell to catalysing various metabolic reactions; from
the transport of macromolecules within and across cells to various recognition and
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immune functions, proteins play vital roles in our bodies and are regarded as the
actual workhorses of the cells. Proteins undergo various post-translational modica-
tions and move through trafcking pathways before they are ready to take up their
function. However, acquiring their specic three-dimensional structure supersedes
all this because only in their specic structural forms can they undertake the func-
tion they are meant to carry out. In this chapter, we shalldiscuss the cellular machin-
ery responsible for maintaining proteins in their functional state during stress
conditions; specically focusing on the Hsp that assist in the folding and refolding
of misfolded and aggregation prone proteins.
8.2 What Is Stress Response?
The stress response can be dened as our involuntary defense reaction to threaten-
ing conditions. This may occur at multiple levels as a response to a different range
of conditions. At a cellular level, the response to any alarming condition, like a
chronic change in the environment away from normalconditions (which may inter-
fere with the physiological functioning of the cells and cause damage to nucleic
acids and proteins) can be identied as a stress response (Kültz 2004). While at the
organism level our adaptive responses are driven by hormonal changes (Charmandari
etal. 2005), the cellular response to damages occurring ata molecular level involves
a cascade of pathways, and molecules that work in cohort to bring the cells back to
their normal functional state. Various kinds of stress at the cellular level include
oxidative, heat, radiation and nutrient deprivation. The major consequences of these
stress are DNA damage, loss of cellular signalling, protein unfolding, misfolding,
aggregation, proteolysis, cellular necrosis and apoptosis. The cellular stress response
may be ofa protective nature where the cell can defend and restore its normal func-
tioning, or of a destructive nature where the conditions are beyond the cell’s ability
to repair. The type, level, and duration of stressful conditions may ultimately deter-
mine the fate a cell. During stress conditions, proteins are misfolded due to changes
in the overall energy landscape. This causes loss of proteinfunction and the accu-
mulation of misfolded proteins in the form of toxic aggregates. The protective stress
response for proteins includes pathways of the heat shock response and the unfolded
protein response (UPR); the destructive response pathways include apoptosis,
necrosis or autophagy (Fulda etal. 2010). The DNA damage response consists of
multiple, complex pathways that restore genomic integrity. Theseinclude the base
excision repair, nucleotide excision repair, and non-homologous end joining
(Kourtis and Tavernarakis 2011). The oxidative stress response helps the cell cope
with the reactive oxygen species, maintain redox homeostasis; and a number of
enzymes like superoxide dismutase and non-enzymatic antioxidants are involved
(Trachootham etal. 2008). In this chapter, we shall mainly focus on the diverse
mechanisms governing heat shock response and the various factors that are involved
in mediating such a response.
8 Molecular Chaperones in Cellular Stress Response
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8.3 Heat Shock Response
The heat shock response was one of the earliest explored stress response mecha-
nisms that wasinitially observed in Drosophila as changes in pufng patterns of
salivary gland chromosomes (Ritossa 1996), followed by changes in gene expres-
sion patterns after heat treatment (Tissiéres etal. 1974; Hightower 1991). The heat
shock response imparts thermo-tolerance to the cells and protects them when they
are stressed due to prolonged exposure to heat. This response is activated by the rise
of a few degreesin temperature from the normal dwelling temperature of the organ-
ism. The regular transcription and translation processes in the cells are halted during
heat shock response, and specic transcription factors (HSFs) which selectively
enhance expression of a set of proteins having protective functions are activated.
However, even during normal conditions these HSFs play important roles in differ-
entiation and development of the organisms (Morimoto etal. 1996). The HSF1
predominantly regulates the heat shock responses, and is itself regulated by its
interactions with heat shock proteins HSP70 and HSP90 (Pirkkala etal. 2001). The
ability to activate transcription and bind toDNA are uncoupled in HSF1 imparting
a higher degree of regulation. The HSF1 exists as a monomer or in complex with
Hspunder normal conditions. During heat shock, HSF1 homotrimerizes and under-
goes hyperphosphorylation which leads to its activation (Cotto etal. 1996). These
HSFsfacilitate the overexpression of Hsp by binding to cis acting sequences on the
genome known as heat shock elements (HSEs). HSP are commonly known as
molecular chaperones for their role in assisting proteins to acquire their native struc-
tures. Hsp prevent heat induceddenaturation and aggregation of proteins, facilitate
the proteins to fold, and assist in the refolding of already denatured proteins
(Lindquist 1986). The Hsp play an active role in facilitating the degradation of
proteins that are unable to fold in order to maintainprotein homeostasis and thus
promote cell survival.
8.4 Cellular Components Providing HS Response
Heat shock response in cells is mediated by concerted actions of heat shock factors
(HSF), heat shock elements (HSE) and Hsp. The heat shock factors as described
above are transcription factors activated during a heat shock. Gram negative bacte-
ria E.coli has aspecic sigma factor 32 (σ32), coded by the rpoH gene, which is a
heat shock promoter specic subunit of RNA polymerase. The σ32 is a positive
regulator and issuppressed by DnaJ during normal conditions (Bukau 1993). The
gram-positive bacteria B. subtilis has a negative regulator HrcA which binds to neg-
atively acting CIRCE elements (controlling inverted repeat of chaperone expression).
Folding of HrcA is mediated by GroE chaperones. During heat shock response,
HrcA doesn’t fold due to the unavailability of free GroE chaperones and hence
the negative regulation of Hsp is switched off (Hietakangas and Sistonen 2006).
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A majority of the eukaryotes have eitherone or multiple of the four heat shock fac-
tors HSF1-HSF4; the condition being different in plants. Arabidopsis has 21 genes
encoding various heat shock factors, divided among three classes and 14 different
groups. The plant HSFs are induced and expressed during heat shock (Hietakangas
and Sistonen 2006). Unlike plants, the transcription factors in animals are like
HSF1. They areconstitutively expressedbut functionally repressedduring normal
conditions by HSP70 and HSP90 (Shi etal. 1998). The DNA binding domain of
HSF recognizes the HSE in a major grove of theDNA double helix. HSEs are highly
conserved and contain inverted repeats of nGAAn (e.g. nTTCnnGAAnnTTCn)
(Amin etal. 1988; Akerfelt etal. 2010). There might be multiple HSEs in the pro-
moter region of a heat shock gene and hence multiple HSFs can bind simultane-
ously in a cooperative manner.
Expression of Hsp is invariably upregulated during heat shock irrespective of the
difference in theirmechanism of action,asdiscussed above. Overall, Hsp or molec-
ular chaperones, are one of the seven different classes of proteins to be overex-
pressed during stress response and were the earliest discovered components of the
heat shock response. The second class comprises components of the proteolytic
system which helps inthe clearance of misfolded and aggregated proteins. The pro-
teolytic machinery of the proteasome has a similar structural organization amongst
different organisms differing onlyin their subunit compositions. The protein degra-
dation machinery requires the translocation of misfolded or partially unstruc-
turedintermediatesbetween cytosol, ER and Golgi in eukaryotes and multiple HSP
members coordinating protein folding and degradation in different cellular com-
partments. The third class of proteins help in DNA and RNA repair, like counteract-
ing the heat induced methylation of RNA.The fourth category comprises enzymes
of the metabolic pathways which reorganize the energy supply of the cell. The fth
category includes kinases and transcription factors that further initiate or inhibit
expression cascades to support the stress response. The sixth class of proteins main-
tain the integrity of thecytoskeleton. Transport proteins and membrane-modulating
proteins makeup the seventh class. All these proteins function together to respond
to the heat stress conditions (Richter etal. 2010). However, we will be discussing
the molecular chaperones in detail in the following sections.
The Hsp are divided into multiple families based primarilyon their size. These
different classes of molecular chaperones and their localization in different cellular
compartments provide a great degree of organizational control and distribution of
roles to execute particular functions within the overall cascade of stressresponse.
HSP60, HSP70, HSP90, HSP100, and small HSP are found across all organisms
and are known to have high similarities within their classes among different organ-
isms. One similarity among all chaperones is that they recognize the hydrophobic
surfaces of proteins which areincreasingly exposed among misfolded and unfolded
proteins, and functionto eitherfold the protein (foldases) or mask the hydrophobic
regions to prevent aggregation (holdases). Despite the name suggesting a partic-
ular function, Hsp play signicant roles in multiple stress response pathways in all
organisms, including oxidative stress in all organisms and drought and osmotic
stress in plants. The roles of molecular chaperones have been observedin the correct
8 Molecular Chaperones in Cellular Stress Response
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folding of a newly translated protein, refolding of misfolded proteins, disaggrega-
tion, translocation, and in the degradation of proteins. HSP60, HSP70, and HSP90
are ATP dependent chaperones which actively support protein folding. The small
HSP and other chaperones simply prevent the misfolding of proteins. The heat
shock factors are regulated by molecular chaperones like HSP70 and HSP90,
thusproving their importance in the overall heat shock response of the cell. HSP70
helps in the translocation of proteins to cellular compartments like ER and alsofacil-
itates retrotranslocation (The reverse movement of the protein from ER to the cyto-
sol, where it can be degraded by the proteasomal machinery) during ER associated
degradation. HSP90 clients include many kinases and steroid receptors, which help
in regulating a multitude of functions through signaling pathways. HSP104 is
known to disaggregate proteins and redirect them to correct refolding pathways.
The widespread presence of chaperones in almost all cellular components where
protein folding occurs, proves that they arekey playersin the heat shock response
machinery. Almost all cellular proteins might have to interact with molecular chap-
erones at least once in their lifetimes, be it during synthesis, folding, targeting, or
degradation.
8.5 Role ofChaperones inMediating Cellular Heat Shock
Response
Cells grow optimally within a narrow range of temperature, pH, and other physio-
logical conditions, but adapt to moderate deviation from such conditions. One of the
most well studied cellular adaptations is the heat shock response (HSR) (Guisbert
etal. 2004). During heat shock conditions, many cellular proteins work to either
rescue the cells from dying, or trigger apoptosis when the damage incurred is irre-
versible. These proteins are referred to as heat shock proteins (Hsp) (Herman and
Gross 2000). Few out of several such Hsp protect proteins from undergoing aggre-
gation, unfold aggregated proteins to make them folding compatible and refold
damaged proteins. These proteins are termed as chaperones (Morimoto etal. 1994).
Molecular chaperone is a major class of protein found at all levels of cellular
organizations ranging from bacteria to humans. They have variable organization
and function depending on the cellular location and complexity of the organism.
Bacterial chaperone proteins are found only in the cytosol as they are not
compartmentalized, but in case of higher organisms, these are also localized in
mitochondria, endoplasmic reticulum, and chloroplasts. Structural and functional
organizations of chaperones are evolutionally conserved within the same kingdom
but vary between them. On the basis of gross molecular weight, the major chaper-
one are classied as: HSP60 (GroEL/GroES, Cpn60/Cpn10, HSP60/HSP10, Tric/
CCT, Thermosome), HSP70 (DnaK, DnaJ, GrpE, HSP70), HSP90 (HSP90, TRAP,
HtpG), HSP100 (ClpA, ClpB, ClpP), sHSP and Trigger Factor (Georgopoulous
et al. 1994; Gross 1996). The other important chaperone proteins playing a role
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in heat shock are Prefoldin, Calnexin / Calreticulin, GRP94, GRP170, AAA
ATPasesPPIases, PDIases, NAC (Nascent polypeptide Associated Complex), CasA
and HtpX (Rani etal. 2016).
8.5.1 Small Heat Shock Proteins (sHsp)
Most organisms have a well-developed sHSP system, which help in their protection
from the thermal, osmotic and salt stresses (Jakob etal. 1993). sHsp have subunit
molecular masses of 12–43kDa. The common feature of all sHSP is the presence of
a highly conserved stretch of 80–100 amino acids in their C-terminus termed as the
“α-crystallin domain” (ACD). It is anked on both side by less conserved N-terminal
domain (NTD) and a C-terminal extension (Kappé etal. 2003; Franck etal. 2004;
Kriehuber etal. 2010). In E.coli major sHsp are IbpA and IbpB.Under normal cel-
lular conditions they help in aggregation prevention and folding of substrate pro-
teins in an ATP independent manner. During stress conditions, along with
anincreased expression, these proteins undergo drastic conformational rearrange-
ments in order to bind to the misfolded proteins and prevent cellular aggregation
(Mani etal. 2016). HSP31 of E.coli functions as a holding chaperone. It cooperates
with the DnaK-DnaJ-GrpE system in managing protein misfolding during stress
conditions (Mujacic et al. 2004). In the pathogenic Halobacterium sp., sHSP1,
HSP-5 and sHSP2 impart protection from thermal stress, solar radiation and high
saltconcentration (Vanghele and Ganea 2010). HSP20 of Mycobacterium tubercu-
losis protects them from macrophage induced stress response and helps in solubili-
zation of heat induced aggregates (Vanghele and Ganea 2010).
In yeast, sHsp HSP26 and HSP42 together, add an additional layer of protection
against a cellular assault like heat shock. Both HSP26 and HSP42 are poorly
expressed during exponential growth, but their expression increases 10-fold under
heat stress suggesting the dominant role they play in a thermally stressed cell
(Haslbeck etal. 2004a). HSP26 exists as a 24-mer under normal conditions, acts
like a holdase for damaged or misfolded proteins and transfers client proteins to the
HSP70 chaperone machinery during heat shock (Haslbeck etal. 2004a, b; 1999).
During heat shock, the 24-mer of HSP26 gets reversibly dissociated into dimers.
This dimeric form then interacts with the unfolded polypeptides and eventually
forms a larger complex, to be presented to chaperones capable of folding the sub-
strate (Stromer etal. 2004). HSP26 also interacts with aggregated proteins, making
them accessible to the HSP104 chaperone (Glover and Lindquist 1998). HSP42
oligomer is a symmetric assembly of dimers organized into two hexameric rings.
HSP42 binds with 30% of total yeast cytosolic proteins (Haslbeck etal. 2004a). It
is a more effective chaperone than HSP26, as ahigher HSP26 to substrate ratio is
needed to prevent aggregation (Haslbeck etal. 2004a). HSP12 exhibits low sequence
homology to the sHSP superfamily and is structurally and functionally distinct, as
it exists exclusively as a monomer (Welker etal. 2010). Liketheother sHsp, HSP12
8 Molecular Chaperones in Cellular Stress Response
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is weakly expressed in exponentially growing cells but overexpressed (100-fold)
during heat shock (Welker etal. 2010).
In plants, there are distinct gene families for sHSP found in different organelles
and a total of 6 families have been classied. The HSP17.6 and HSP17.9 reside in
the cytoplasm, HSP21in the chloroplast, HSP22in the ER, HSP23in the mitochon-
dria and HSP22.3in the cell membrane (Wang etal. 2004). They have been sug-
gested to be involvedeither in maintaining the structure of a heat stressed cell
(Lindquist and Craig 1988) or to protect the photosynthesis machinery during heat
shock (Schuster etal. 1988; Chen et al. 1990). Genetically modied plants with
higher thermo-tolerance have been designed by constitutive upregulation of
sHSP.For example, the HSF3 gene of Arabidopsis thaliana was modied to express
it in non-heat shock conditions andwas shown toincrease the basal thermotolerance
of the plant (Prändl et al. 1998). Overexpression of HSP17.6 results in a higher
tolerance to drought and salinity in Arabidopsis thaliana, while the natural elevated
gene expression is observed in case of heat shock (Sun etal. 2001).
In humans, Group I sHsp consists of HSP27, HSP20, HSP22, and αB-crystallin.
They are found in various tissues and are heat inducible. Group II sHsp consists of
HSPB9, HSPB10, HSPB3, HSPB2 and α-crystallin, and they are involved in cell
differentiation and are restricted to certain tissues (Taylor and Benjamin 2005).
HSP27 forms oligomeric species when subjected to higher temperatures and this
results in increased chaperone activity (Bakthisaran etal. 2015). They predomi-
nantly function as ‘holdases’,keeping the substrates in a folding competent globular
state that can later be presented to ‘foldases’ like HSP60/10 or HSP70 to make them
functional (Eyles and Gierasch 2010). Under conditions of heat stress, they also
prevent aggregation by binding to late unfolding intermediates and keep them in a
stable, soluble complex. sHSP may also be involved in the transient/reversible reac-
tivation of early unfolded intermediates and this process may be ATP dependent,
although no ATP hydrolysis has been observed (Rajaraman etal. 2001). ATP bind-
ing is thought to trigger a conformational change that aides Hsp to release their
refolding-competent substrates (Muchowski etal. 1999).
8.5.2 The Chaperonin System (HSP60)
Chaperonins are double ring complexes of 800–900kDa which help in the folding
of many cellular proteins under normal and stress conditions (Spiess etal. 2004;
Hemmingsen etal. 1988; Vabulas etal. 2010). These are further classied as group
I and group II chaperonins (Horwich etal. 2007).
In bacteria and symbiotic organelles like mitochondria and chloroplasts, Group I
chaperonins (cpn60) are found. These chaperonins are termed as GroEL in E.coli,
mtHsp60in mitochondria and Rubisco binding protein in chloroplast (Figueiredo
etal. 2004). These require co-chaperonin GroESor HSP10 to functionin prokary-
otic and eukaryotic cells respectively. Under normal cellular conditions, GroEL/ES
is constitutively expressed in bacterial cytoplasm and helps in the folding of
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substrate proteins in an ATP dependent fashion. Under environmental stress condi-
tions, the expression of these proteins increase by 15–20% (Georgopoulos etal.
1994; Gross 1996)and also leads to a few structural and functional modications of
the chaperone system, which enable them to fold or hold the aggregation prone
proteins. One such modication is the phosphorylation of the double ring which
mediates substrate folding in an ATP independent manner (Sherman and Goldberg
1994). GroEL also acts as a holding chamber for substrate proteins during thermal
stress and helps them regain their folding function once normality is restored
(Carrascosa etal. 1998). Mitochondrial Hsp60, with the help of its co-chaperonin
HSP10 helps in thefolding and assembly of imported proteins inside the matrix of
mitochondria. In HSP60 conditional mutants, aggregatesaccumulate andare unable
to assemble into functional complexes. The plastid HSP60 chaperonin found in
chloroplasts consists of two different subunits which make them different from
other members of the HSP60 family (Levy-Rimler etal. 2002). These two distinct
subunits known as CPN60α and CPN60β share about 50% sequence identity
(Boston etal. 1996). The tetradecameric structure consists of α7β7oligomers where
the assembly of α7 is dependent on the β7 homo-oligomer, thus forming two homo-
geneous rings, and the oligomers having a seven fold symmetry (Waldmann etal.
1995). The chloroplast co-chaperonin can bind to both GroEL and ch-CPN60 and
assists in thefolding of proteins in both cases. Its structure however, is markedly
different from GroES and contains two GroES like domains connected by a short
linker region. Some reports have conrmed the presence of 10kDa CPN10 and
20 kDa dimer like CPN20 existing simultaneously in the chloroplasts (Hill and
Hemmingsen 2001; Levy-Rimler etal. 2002).
Group II chaperonins are found in archaea (Thermosome) andthe cytoplasm of
eukaryotes (TCP/CCT) (Ditzel etal. 1998; Leitner etal. 2012). They do not require
the co-factorHSP10 as they have specialized α-helical extensions in their apical
domain that function as a built-in lid (Vabulaset al. 2010). Archaeal Group II chap-
eronins (Thermosome) consists of two stacked octameric rings with two different
kinds of subunits α & β (Klumpp etal. 1997). The mechanism of folding of non-
native protein substrates is similar to GroEL andinvolves binding of the substrate at
the apical domain, followed by ATP hydrolysis and release of thefolded substrate
fromthe cavity (Lopez etal. 2016). During stress conditions, Group II chaperonins
form a large octadecameric β complex which is more efcient insubstrate binding.
It also helps in membrane stabilization of archaeal cells during stress conditions
(Chaston etal. 2016). The group II chaperonins in the eukaryotic cytosol are known
as TRiC (TCP-1 ring complex) or CCT (chaperonin containing TCP-1) and like
their archaeal counterparts have eight or nine rings, each containing eight paralo-
gous subunits (Frydman 2001). The general domain structure of the group II chap-
eronins is akin to GroEL (Ditzel et al. 1998). The closing and opening of these
segments to encapsulate the substrate in the TriC/CCT cavity is ATP dependent
(Meyer etal.2003a). TriCs interact functionally with the co-chaperone prefoldin
(Vainberg etal. 1998; Siegert etal. 2000) and HSP70 (Langer etal. 1992), which
serve to transfer substrates to this chaperonin.
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8.5.3 HSP70
HSP70 family is involved in a multitude of functions in all organisms and are found
in various cellular compartments. HSP70 facilitates translocation, protein import,
and signal transduction along with assisting in therefolding of substrate proteins
and preventing their aggregation (Frydman 2001; Miemyk 2017). HSP70 consists
of two domains, a highly conserved 44kDa N-terminal ATP binding domain, and a
15kDa C-terminal peptide binding domain (PBD) which consists of a β-sandwich
motif and an α-helical lid segment (Vabulas etal. 2010; Yu etal. 2015). In eukary-
otes, under normal conditions, HSP70 exist as complexes with either HSP90 or
co-chaperones like HSP40 and others, and is known as Heat Shock Cognate 70
(HSC70). The constitutively expressed HSC70 assistin the folding and transloca-
tion of newly synthesized proteins during normal conditions (Hartl etal. 2011).
Under heat stress conditions, the stress-inducible HSP70 isoforms are over-
expressed. This is achieved by itsinteraction with HSF, which transcriptionally
activates several heat shock genes including that of HSP70. HSP70s function is
generally conserved in all organisms (Akerfelt etal. 2010). Nascent polypeptides
form the bulk of its clients and undergo chaperoning via the ATP-dependent reac-
tion cycle of HSP70 which is regulated by HSP40 and nucleotide-exchange factors
(NEF) (Kampinga and Craig 2010; Mayer 2010). The β-sandwich motif of the PBD
recognizes an extended, seven-residue hydrophobic patch of an aggregation prone
protein, especiallywhen they are locally surrounded by positively charged residues
(Rudiger etal. 1997). The binding of the peptide is ATP-dependent and is regulated
by a conformational change in the β-sandwich motif (Mayer 2010). In the ATP-
bound state, the lid adopts an open conformation resulting in a high binding afnity
for the peptide. Hydrolysis of ATP facilitates lid closure and is accelerated by
HSP40, leading to the peptide getting locked inside. Following the hydrolysis of
ATP, NEF binds to the ATPase domain and catalyses ADP-ATP exchange that result
in the opening of the lid and release of the substrate, presumably in their non-native
conformation. These intermediates might then undergo multiple rounds of binding
and release till they acquire their native conformation (Hartl etal. 2011). Under
stress conditions (apart from folding nascent polypeptides) HSP70 prevents aggre-
gation of non-native substrates by transiently shielding exposed hydrophobic seg-
ments and keeping them in a folding competent state, which may subsequently be
transferred to the chaperonin cage for completion of the folding process (Vabulas
etal. 2010). BiP, the HSP70 paralog in the ER, binds to unfolded regions of the
protein manifesting exposed hydrophobic residues and has an ATP dependent
mechanism of substrate binding and release, similar to its cytosolic counterpart. BiP
plays an active role in the Unfolded Protein Response pathway (UPR) and in ER
Associated Degradation (ERAD), both of which occur when misfolded proteins
start accumulating in ER.It does so by interacting with several co-chaperones that
assist in protein folding and quality control. One of them is Erdj3, an HSP40
paralog in the ER, which interacts with partially folded intermediates and presents
them to BiP.Another cohort is BAP, a NEF that plays a similar role as its cytosolic
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counterparts, promoting ADP release and facilitating BiPs transition to the open
state (Ma and Hendershot 2004).
In yeast, SSA and SSB are subclasses of cytosolic Hsp70 that are constitutively
expressed, while SSA3 and SSA4 are known to be induced upon exposure toheat
shock (Santoro etal. 1998). Interaction with the chaperone Ydj1 (Hsp40), a homo-
logue of the bacterial DnaJ protein, accelerates the ATPase activity of Hsp70. KAR2
(ER Hsp70) is a yeast homolog of BiP protein which gets induced within 10minutes
of heat shock (Normington etal. 1989). Kar2 is responsible for the translocation of
proteins across the ER membrane through co-translational as well as post-transla-
tional pathways (Brodsky etal. 1995). It is also involved in theretrograde transport
of defective non-functional substrates from the ER to thecytosol for proteosomal
degradation via ERAD, and thereby contributes to ER proteostasis (McCracken and
Brodsky 1996). The promoter region of the Kar2 gene contain unfolded protein
response elements (UPREs), and itgets induced when unfolded polypeptides start
accumulating in the ER lumen (Cox and Walter 1996). As is the case for human
cytosolic Hsp70, Kar 2 interacts with co-chaperonesHsp40/J proteins (Sec63, Scj1,
and Jem1) and nucleotide exchange factors (Sil1) (Nishikawa and Endo 1997;
Sadler etal. 1989; Schlenstedt etal. 1995). Sec63 acts as a Kar2 ATPase activator
(Lyman and Schekman 1995), while Scj1 functions to counter the misfolding of
proteins occurring due to lack of a carbohydrate modication (Silberstein etal. 1998).
SSC1 and SSC 3 are the two mitochondrial Hsp70 chaperones which are involved
in the heat shock response (Wagner etal. 1994). Binding of the SSC1 to the unfolded
proteinis critical for post-stress(Baumann etal. 2000). Mdj1 (J protein),the yeast
mitochondrial HSP70 cofactor, helps in preventing heat induced protein aggrega-
tion in mitochondria (Rowley etal. 1994). It is also involved in protein folding.
Furthermore, in addition to folding, its interaction with SSC1 alsofacilitates the
clearance of misfolded mitochondrial proteins. MGE1 is the only mitochondrial
NEF known to interact with, and therefore assist the Ssc1 chaperone action by pro-
moting the release of bound nucleotide (Wagner etal. 1994).
The HSP70 family in plants has been subdivided into 4 subgroups based on
C-terminal sequence motifs and their cellular localization. The cytosolic HSP70
contains the EEVD motif; the ER HSP70 contains the HDEL motif, the HSP70
molecules found in plastids havethePEGDVIDADFTDSK motifand those in mito-
chondria have aconserved PEAEYEEAKK motif (Guy and Li 1998). These motifs,
known as anchors, are identied by co-chaperones and are involved in substrate
binding through theassistance of co-chaperones (Freeman etal. 1995). Plant cells
are different in that they contain multiple(2-5) HSP70 family members within the
ER (Ray etal. 2016). In Arabidopsis, the totalHSP70 family members are encoded
by about 18 genes, (Lin etal. 2001)and about 12 genes of the HSP70 family have
been identied in the spinach genome (Guy and Li 1998). This gives an idea about
the functional diversity of this chaperone family (Wang etal. 2004). Mechanisms by
which HSP70 mediates its cellular function have been difcult to study in plants
due to the poor survival of deletion mutants and the unavailability of efcient
inhibitors (Sarkar etal. 2013). In prokaryotes, the DnaK/DnaJ system belongs to
HSP70/HSP40 family of heat shock proteins. They function with the prokaryotic
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NEF GrpE.DnaK is an ATP-dependent chaperone which requires DnaJ and GrpE
for substrate binding through its ATPase cycle. During stress conditions, DnaJ
undergoes certain functional modications which stimulates the ATPase activity of
DnaK (~500 fold). During stress conditions, DnaK/DnaJproteins also interact with
ClpB and reactivate the inactivated proteins (Liberek et al. 1992). DnaK/DnaJ
system is absent in archaeal species except for a few halophiles, hence may not have
any particular role in alleviating archaeal stress. The functional analogs of DnaK/
DnaJ system in archaea are prefoldin and GimC protein (Rani etal. 2016).
8.5.4 HSP90
HSP90 is another major player that functions downstream of HSP70in the confor-
mational maturation and functional activation of several important classes of pro-
teins that actively take part in various cell signaling pathways. While the basal
HSP90 content is about 1% of the total cellular proteincontent, it may increase to
4–6% during the stress response (Young etal. 2001; Wegele etal. 2004). HSP90 is
usually present in the cytosol, but they may also be found in the ER, mitochondria
and plastids (Krishna and Gloor 2001). HSP90 functions as a dimer; the monomer
units covalently joined at their C-terminal domains via the dimerization domain.
The N-terminal domain binds and hydrolyzes ATP and is joined to the C-terminal
domain by a middle domain, which participates in substrate and co-chaperone bind-
ing. In all organisms HSP90 acts as a scaffold for several if not all protein folding
pathways and components. HSP90 dimer undergoes an ATP driven reaction cycle of
substrate binding and release achieved by the transition from an open nucleotide-
free to a closed ATP-bound state ‘committed to hydrolysis. HSP90 works in cohort
with multiple co-chaperones like HSP70,HOP, the J-domain proteins (HSP40) and
p23. These co-chaperones mediate the interactions between HSP90 and the sub-
strates in most of the cases (Li etal. 2012).
In humans, clients of this ~ 90Kda chaperone number over 200, which includes
kinases, nuclear receptors, transcription factors, telomerase and many other proteins
(Zhao etal. 2005; McClellan etal. 2007). Under stress conditions HSP90α is over-
expressed and shows marked increase in interactions with certain clients. HSP90α
has a higher potential for oligomerization and undergoes temperature-dependent
oligomerization above 45°C, and denatured substrates like DHFR bind specically
with these higher order oligomers (4-mer, 6-mer, and 8-mer). Oligomeric forms of
HSP90 display manifold higher afnity for denatured substrates as they themselves
undergo ‘unfolding', wheretheir hydrophobic patches are exposed (Csermely etal.
1998). Like HSP70, HSP90 prevents irreversible denaturation of substrates like
luciferase and can act as ‘holdases’. When cells go back to the resting phase, these
substrates can be captured by ‘foldases’ and reactivated (Minami and Minami
1999). Tumour Necrosis Factor Receptor-Associated protein 1 is the mitochondrial
homologue of Hsp90 (Felts etal. 2000). It is a ~75kDa protein (Chen etal. 1996)
which is structurally similar to its cytosolic counterpart, except the absence of an
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MEEVD motif in the C-terminus that binds to co-chaperones like p23, indicating
that the regulation of TRAP-1 might be orchestrated by a different set of co-
chaperones in a yet unknown fashion (Felts etal. 2000). TRAP-1 functionally dif-
fers from its ER counterpart, GRP94in that it adopts a closed conformation upon
ATP binding, but this alone is insufcient for commitment towards ATP hydrolysis
as the propensity to adopt an open conrmation even before ATP hydrolysis is
greater than its ATP hydrolysis rate. This kinetic partitioning essentially reduces the
turnover number of ATP and signies that the dissociation of ATP is favoured to its
ATPase activity (Leskovar etal. 2008). Under heat shock conditions, its ATPase
activity increases by ~ 200 fold (Altieri etal. 2012). Although downstream targets
involved in this process is not fully understood, it is probably the chaperone
Cyclophilin D (a mitochondrial matrix PPIase), a key component of the Permeability
Transition Pore complex (PTP). Opening of the PTP is known to induce mitochon-
drial apoptosis, and by refolding Cyclophilin D in a closed PTP conguration
through its ATPase activity, TRAP-1 is perhaps able to mediate cell survival (Green
and Kroemer 2004; Kang et al. 2007). The proteins classied in HSP90 family
range from 80–94kDa in size and about 70% sequence identity has been observed
between the plant and other eukaryotic counterparts of HSP90 (Lindquist and Craig
1988). The HSP90 family in Arabidopsis has seven members where AtHSP90–1 to
AtHSP90–4 are present in the cytoplasm, AtHSP90–5 is present in the plastids,
AtHSP90–6 in the mitochondria and AtHSP90–7 in the ER (Krishna and Gloor
2001; Wang etal. 2004).It has been observed in Arabidopsis that HSP90 is involved
in mediating the stress response pathways through its interactions with the heat
shock factor (HSF) (Yamada etal. 2007). Overall, studiescarried out in plants sug-
gest important roles of HSP90 in many aspects of plant development and stress
response. HtpG is the Escherichia coli homolog of HSP90. It normally acts as a
holder chaperone that transiently maintains the nascent protein in a conformation
accessible to DnaK. During stress conditions, its expression increases 5–10-fold
and it binds to aggregated proteins with the help of ClpB and re-presents them to the
DnaK/DnaJ system (Thomas and Baneyx 2000). In yeast, HSP90 chaperone is
encoded by the Hsp82 gene, which is overexpressed under heat shock conditions
(Smith etal. 1991). Yeast HSP90 chaperone system is also involved in overcoming
the deleterious impact of heat shock on the cell surface (Imahi & Yahara 2000).
8.5.5 HSP100/Clp
The HSP100 family is primarily known for its unique function of remodeling pro-
tein complexes and disassembling protein aggregates, facilitating either refolding or
degradation of the aggregated proteins (Goloubinoff etal. 1999). The HSP100 are
hexameric ring structures belonging to AAA+ ATPase family (Burton and Baker
2005) with two dened classes based on distinct N and C-domains. The class Ipro-
teins are ClpA, B, C, D, E having two AAA modules, whereas the class II proteins
ClpM, N, X, and Y have only one AAA module (Lee etal. 2007). In E.coli the major
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HSP100 are ClpA, ClpB, ClpC, ClpX and ClpY.They help in thedisassembly of
oligomers and aggregates during stress (Smith etal. 1999). HSP104 is the yeast
homolog of ClpB. It recognizes misfolded proteins within an aggregate, unfolds
them and ultimately delivers the substrates into various refolding pathways
(Schirmer etal. 1996). Together with HSP70 and HSP40, it resolubilizes and refolds
the substrates. HSP78 (a yeast mitochondrial aggregase), plays a role in the reacti-
vation of proteins damaged due to stress, thus imaprting thermotolerance (Janowsky
etal. 2006). It binds to misfolded polypeptides in the matrix and stabilizes them,
therebypreventing their aggregation (Schmitt etal. 1995). One of the major roles of
HSP78 is resolubilization of the Ssc1 (mtHsp70) chaperone which itself tends to
misfold during stress (Sichting etal. 2005). HEP1 (mtHsp70 escort protein) and
Pim1 (involved in mitochondrial proteolysis) are some of the other proteins in mito-
chondria which are involved in HSR (Sichting et al. 2005; Wagner etal. 1994).
HEP1 plays a complementary role to Hsp78 in maintaining functional SSC1
(Sichting etal. 2005). It assists Ssc1 to maintain its solubility and function during
stress (Sichting etal. 2005). Multiple HSP100 membersexist in Arabidopsis; four
ClpB, two ClpC, and one ClpD.Although they are present at basal levels in cells
under normal conditions, an increased expression is observed during heat stress.
The cytosolic ClpB1 of Arabidopsis, known as AtHSP101 is present in high levels
during heat stress and imparts thermotolerance to plants (Glover and Lindquist
1998; Lee etal. 2007).
8.5.6 Other Chaperones
Many other chaperones also play a role in stress tolerance; e.g., PPIases, AAA
ATPases and so on. PPIase proteins include cyclophilins, FKBPs and parvulins
(Maruyama etal. 2004). They help in cis-trans prolineisomerization in a polypep-
tide chain and help in the fast folding of kinetically trapped proteins. AAA ATPases
are mainly found in archaea and eukaryotes. The major AAA ATPase of archaea is
CDC48 and AMA which function as major proteasomal ATPases. These proteins
regulate proteasomal protein degradation in archaea (Forouzan etal. 2012). In yeast
and humans, most proteins that are translocated into the ER are N-glycosylated with
a branched glucose-3-mannose-9-N-acetylglucosamine-2 (Glc3Man9) glycan chain
(Helenius and Aebi 2004). This oligosaccharide moiety serves as a recognition sig-
nal for lectin-like chaperones calnexin (CNX) and calreticulin (CRT) (Daniels etal.
2003). CRT prevents thermal aggregation and promotes recovery of nonglycosyl-
ated substrates. This happens due to enhanced polypeptide binding property of CRT
under heat stress. CRT also forms oligomers under heat stress via certain conforma-
tional changes that occur in its C-terminal acidic domain. Oligomerization of CRT
and enhanced polypeptide binding are concomitant and explain how CRT acts as a
chaperone. CNX may act to recruit other chaperones like the PDI family member
Erp57 that catalyses disulphide bond formation in a highly oxidized ER lumen or it
may even act as a chaperone, binding to exposed polypeptide stretches of misfolded
AU1
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glycoproteins. Both these functions of CNX are enhanced under heat stress condi-
tions. However, these chaperones may also ‘generally’ bind to glycoproteins inde-
pendent of their folding state, suggesting that there is no specic recognition of the
attached polypeptide chain (Buchberger etal. 2010). Other chaperones like GRP94,
Peptidyl-ProlylIsomerases (PPIase) and GRP170 along with the ones mentioned
above form a large ER-localized multi-protein complex that functions as a network
and bind to unfolded proteins in the ER rather than existing as free pools that get
individually assembled onto protein clients. Grp94 most likely acts as a scaffold like
its cytosolic HSP90 counterpart and forms an important part of this multi-chaperone
complex (Ma and Hendershot 2004). Table8.1 lists the major chaperones involved
in attenuating the adverse effects of heat shock and their subcellular localization.
8.6 Detailed Mechanism ofChaperone Assisted
Protein Folding
Out of the many chaperone systems described briey in the previous section, our
group has mainly focussed on a couple of chaperone systems namely GroEL/ES (E.
coli) and HSP90 (human). Based on the various structural and functional studies
carried out by our group since the last decade on the chaperonin GroEL/ES system,
we have better understood the importance of this system in aggregation prevention
of denatured substrates, refolding of substrates and survival of E. coli. While we
have only recently started working on the human HSP90 system, we have obtained
novel information regarding the contribution of HSP90s structure in modulating its
chaperone properties using certain HSP90 inhibitors. The following section
describes in detail the structure and function of GroEL/ES and HSP90 during nor-
mal and under stress conditions.
Table 8.1 Major Chaperones described herein and their subcellular localization (Modied from
(Graner etal. 2014))
Subcellular localization
Major chaperones ER Mitochondria Cytosol Nucleus Cell Surfacea
HSP27 ✓ ✓ ✓
HSP60 ✓ ✓ ✓
HSP70/HSC70 ✓ ✓ ✓
GRP78 (BiP) ✓ ✓ ✓ ✓ ✓
HSP90 ✓ ✓ ✓
HSP110 ✓ ✓ ✓
GRP94 (gp96) ✓ ✓ ✓
CNX/CALRb✓ ✓ ✓
PDIc✓ ✓
aCell surface localization is mostly associated with tumour cell surfaces
bCalnexin/Calreticulin
cProlyl Disulphide Isomerase
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8.7 GroEL/ES Mediated Protein Folding
8.7.1 GroEL/ES Structure
GroEL in its normal state is a porous cylindrical protein made of 14 subunits
arranged in nearly 7- fold rotationally symmetrical rings stacked back to back, and
forms a cage like structure with a central cavity (Braig etal. 1994). Each GroEL
subunit further folds into three domains (Braig etal. 1994; 1995). The apical domain
(residues 190–345) whichis rich in hydrophobic residues and acts as a binding site
for the non-native substrates and co-chaperonin GroES (Fenton etal. 1994). The
intermediate domain (residues 134–190, 377–408) acts like a hinge between the
apical and equatorial domains. This also helps in allosteric communication between
the two domains. The equatorial domain consists of sub-domains E1 (residues
4–133) and E2 (residues 409–523) that form all the intra and inter-subunit interac-
tions required for the proper folding of the monomeric subunit and their assembly
into the tetra-decameric form (Braig etal. 1994, Hayer-Hartl etal. 2016). It also
houses the nucleotide binding pocket since GroEL works in ATP-dependent manner
(Braig etal. 1994). GroES is a single seven membered ring with identical subunits
of 10kDa that binds to one or both ends of the GroEL cylinder in presence of a
nucleotide. Each subunit consists of a β-barrel body with an extended hydrophobic
mobile loop that interacts with the apical domain of GroEL and providesan enclosed
cavity for the folding of substrate proteins (Braig etal. 1994, Fenton et al. 1994;
Hendrick and Hartl 1993). Binding of GroES to the GroEL cylinder doubles the
volume of central cavity to provide sufcient space for the folding of substrate
proteins. Under normal conditions, GroEL interacts with non-native substrates
post- translationally (Fenton etal. 1997), while native proteins under stress are sub-
jected to unfolding that leads to the formation of intermediate ‘aggregation prone’
states that remain bound to GroEL.These can thenbe refolded back to their native
form, presented to other chaperones, or taken up by the proteolysis machinery
(Hartl etal. 2011).
8.7.2 GroEL Mechanism ofSubstrate Folding underNormal
Conditions
GroEL helps in the folding of about 5–15% of total cellular protein under normal
conditions (Ewalt etal. 1997). Depending upon the size of thesubstrate protein, it
assists in folding in two different ways: (I) Cis- mechanism of folding and(II)
Trans- mechanism of folding. The GroEL cavity can encapsulate substrate proteins
ranging between 54–57kDa (Sakikawa etal. 1999) and help in their folding by
providing an Annsen cage inside the cavity. This is termed as the cis-mechanism
of substrate folding. GroEL is not able to encapsulate large proteins (> 60 kDa)inside
its cavity and they can undergo multiple rounds of binding and release at the apical
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domain of GroEL before reaching their nal folded or ‘committed to folding’ state.
Both GroES and ATP are required to release the folded/partially folded substrate
from the cis-ring (Fenton and Horwich 1997; Dahiya and Chaudhuri 2014).
8.7.3 Cis- Mechanism ofGroEL Action
The asymmetric complex of GroEL-GroES is the most common form found in the
cellular milieu, along with a few symmetric complexes of GroEL (Weissman etal.
1995). Afew other reports however, show that both types of complexes can exist in
equimolar concentration in cellular milieu and that the ratio of symmetric to the
asymmetric complex is an ADP dependent phenomenon (Yang etal. 2013; Lizuka
and Funatsu 2016). The asymmetric complex has one ring of GroEL occupied by
GroES, while another ring remains ready to accept a non-native substrate protein.
Due to the presence of hydrophobic residues at the apical domain, newly synthe-
sised polypeptides or partially folded substrates bind to the empty GroEL ring. The
binding of substrate protein leads to a conformational change in the same ring which
promotes subsequent binding of GroES and ATP.This leads to the formation of an
isolated cage for the folding of thesubstrate, known as the cis-ring. The conforma-
tional changes that occur during the binding of GroES and ATP make the GroEL
cavity hydrophilic, which in turn providesa proper environment for the folding of
substrate protein. ATP hydrolysis is a slow process with one molecule of ATP
hydrolysed in 8–10seconds. The hydrolysis of ATP lowers the binding afnity of
GroES to the apical domain. This releases GroES and the folded substrate from the
GroEL cis ring; simultaneously ATP binds to the opposite ring along with another
substrate and a new cycle is initiated. (Fenton and Horwich 1997; Weissman etal.
1995). Recent reports show that both rings of GroEL act in a concomitant fashion
during the folding process (Yang etal. 2013). The following schematic explains the
mechanism of cis- folding (Fig.8.1).
8.7.4 Trans Mechanism ofGroEL
Large proteins with molecular weight (>60kDa) are too big to t inside the GroEL
cavity. This does not involve cis-encapsulation, but requires GroES binding to the
trans ring to release either folded or partially folded substrates (Chaudhuri etal.
2001). As thecis-ring binds with the substrate protein, the trans ring acts as a bind-
ing site for GroES, so this mechanism is termed as folding in-trans. There are many
substrate proteins, e.g., 70kDa tail spike protein of phage p22, 86kDa α/β heterodi-
mer, 82kDa mitochondrial aconitase, dimeric citrate synthase, etc. that fold via this
mechanism (Weissman etal. 1995, Chaudhuri etal. 2001). In this process, the non-
native substrate binds to the apical domain of theasymmetric GroEL-GroES com-
plex, and undergoes folding with domain rearrangements or prevents aggregation
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(Chaudhuri etal. 2001). Theapical domain of GroEL can also bind with the ‘burst
phase intermediate’ state of substrate proteins. To prove this hypothesis, experi-
ments were carried out using slow folding Malate Synthase G (MSG), a 89kDa
multi-domain monomeric protein. Our observations suggest that binding of MSG to
GroEL accelerates the slowest kinetic phase of the spontaneous protein folding
pathway. Due to the large size of thesubstrate, GroES is not able to bind to this ring,
but ATP hydrolysis continues, which helps in the release of the folded substrate
from the ring. Sometimes the release of thesubstrate depends on the binding of both
GroES and ATP to the opposite ring. The binding of GroES and ATP to the opposite
ring also accelerates the release of folded/partially folded substrate from the GroEL
ring (Paul etal. 2007; Dahiya and Chaudhuri 2014). The trans mechanism of sub-
strate folding occurs under normal cellular conditions and also during thermal
stress. It helps in preventing the irreversible aggregation of thermally denatured
proteins. Study of the thermal unfolding pathway of citrate synthase (CS) show that
CS unfolds via an inactive dimeric intermediate. Further unfolding of these interme-
diates led totheir irreversible aggregation. GroEL interacts with this dimeric unfold-
ing intermediate, dissociating them into monomers which stably associate with
GroEL (Grallert etal. 1998). The following schematic explains the mechanism of
Trans-folding (Fig.8.2).
Fig. 8.1 Proposed model for GroEL/ES assisted folding of small proteins via cis- mechanism):
(I) Open ring of GroEL–GroES–ADP complex acts as an acceptor state for the non-native poly-
peptide. (II) Binding of GroES to the GroEL–ATP complex leads to conformational changes in the
apical domain of GroEL; consequently, polypeptide enters in the central cavity. (III) Folding
occurs in the cis cavity before the ATP gets hydrolysed, this weakens the interaction between
GroEL and GroES. (IV) Binding of ATP to the trans ring promotes the release of (N– Native, Ic–
partially folded, U– misfolded) from the cis ring. (V) At the same time, binding of GroES to
GroEL allows GroEL to alternate its rings between binding-active and folding-active states.
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8.7.5 Passive Models ofGroEL-Mediated Folding
GroEL passively suppresses protein aggregation (Pelham 1986; Ellis and van der
Vies 1991; Agard 1993) by binding to the exposed hydrophobic regions of aggrega-
tion prone proteins. GroEL specically recognises and binds with the on-pathway
intermediate states, which shifts the overall folding equilibrium away from aggrega-
tion and provides a pool of folding competent monomers that could reach their
native state by further folding or assembly. In a purely passive folding model, high
concentrations of an aggregating molecule cause non-linear increase in the rate of
aggregation (Van den Berg 1999; Ellis 2001). GroEL prevents the formation of
unfavourable intermediates that could lead to aggregation and hence aid in the
proper folding of its substrates.
Fig. 8.2 Proposed model for GroEL/ES assisted folding of large proteins via trans-
mechanism: The burst phase intermediate of MSG is captured by GroEL (orange colored) to form
GroEL-MSG complex. This binding induces minor structural rearrangements to give rise to a more
folding-compatible state. Further addition of GroES/ATP or ATP releases the GroEL-bound form
of MSG, which folds to the native state via formation of a compact intermediate, that is structurally
quite close to the native MSG.GroES (shown in blue) binds in Trans to the folding polypeptide and
doubles the ATP-dependent reactivation rate
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8.8 GroEL inStress
8.8.1 Synthesis ofGroEL during Stress
During heat shock, activation of σ32 transcription factor leads to an increased
production of Hsp inside E.coli. GroELpopulation increases from a basal level of
1–2% to 10–15% (of the total cellular proteincontent). Non-native substrates bind-
ing to GroEL results in a 2-fold increase (30% of the total cytoplasmic protein
content) in the clientele of GroEL, as compared to normal conditions (10–15%)
(Bukau 1993).
8.8.2 Structural andFunctional Modications
duringHeat Shock
Foldase During heat shock, GroEL undergoes phosphorylation allowing it to
function without GroES. Enhanced ATPase activity in the phosphorylated form
ensures relatively fast release of the folded substrate from GroEL.The folding ef-
ciency of phosphorylated GroEL is 50–100 fold higher than its native counterpart.
Phosphorylation is a reversible process and once the cell is relieved of stress, GroEL
undergoes de-phosphorylation and resumes its normal function inside the cell
(Sherman and Goldberg 1994).
Holdase Our studies on monomeric GroELshow that the apical domain is the most
stable region in the GroEL subunit as it requires 4.0M urea and 70°C to undergo
complete unfolding (Golbik etal. 1998; Puri and Chaudhuri 2017). This is a kind of
adaptation in the GroEL structure that can possibly hold aggregating substratesunder
stress conditions. It is also observed that during thermal stress, GroELs protein fold-
ing activity is reduced and itstarts acting as holdase by binding to the aggregation
prone proteins, thusbehaving as a storehouse of proteins. The molecular basis for
such a functional transition is the loss of inter-ring signalling and negative co-
operativity, whichslows down the release of GroES and the unfolded proteins from
the GroEL cavity. This phenomenon is also reversible with GroEL reverting to its
normal function after heat shock (Llorca etal. 1998).
Unfoldase Strong binding of substrate proteins at the GroEL apical domain can be
the main cause of unfolding or ‘stretching’ of bound substrates. The unfolding
activity occurs through inter-domain movement where stretching of the apical
domain helps in ‘opening up’ of the aggregating substrates. The unfolded substrate
remains bound to the GroEL apical domain during stress conditions, but once cel-
lular conditions become normal, GroEL is able to perform its folding function
(Grallert etal. 1998).
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8.8.3 ATP Independent Aggregation Prevention
byGroEL Protein
Aggregation prevention tendency of GroEL is an ATP independent process. This
proves useful during stress conditions, because of increasing load of aggregating
proteins and relatively more ATP consumption to maintain cellular homeostasis
(Soini, etal. 2005). In vivo studies in bacteria and yeast demonstrated that depletion
of either GroEL or mitochondrial Hsp60 resulted in aggregation of a large number
of newly translated proteins (Cheng etal. 1989; Horwich etal. 1993). Similarly,
many invitro studies demonstrated that GroEL can rapidly and efciently bind
to non-native states of several proteins and arrest their aggregation e.g., malate
dehydrogenase (MDH; Ranson etal. 1995), ribulose-1,5-bisphosphate oxygenase-
carboxylase (RuBisCO; Goloubinoff etal. 1989), etc. Our current study on aggrega-
tion prevention of Maltodextrin glucosidase (MalZ) demonstrate that GroEL can
prevent aggregation of this protein without the involvement of GroES and ATP (Puri
and Chaudhuri 2017). The passive binding of non-native intermediates to GroEL
can prevent their aggregation by disallowing random hydrophobic interactions.
Once captured proteins reach the folding competent state, they must be released
back into thefree solution, to undergo completion of the nal steps of folding or
oligomer assembly without ATP (Lin and Rye 2006).
8.9 HSP90 Mediated Protein Folding
Structure
HSP90 functions as a exible dimer inside the cell; the dimers consist of two mono-
mers joined at their C-terminus via the dimerization domain. The N-terminus consists
of a conserved Bergerat ATP/ADP binding fold that also binds to Geldanamycin and
Radicicol, competitive inhibitors of ATPbinding (Pearl 2016; Bergerat etal. 1997;
Stebbins etal. 1997; Roe etal. 1999). An N-terminus ‘lid’ segment that responds to
ATP binding has been found, consisting of two highly conserved glycine clusters
(Prodromou etal. 2000). The N-terminus is connected to the Middle (M) domain by
a exible linker that has been shown to convey allosteric modulations from the
M-domain and the C-terminus to the N-terminus, resulting in global conforma-
tional changes. The M-domain binds to co-chaperones that “present” client pro-
teins to HSP90. The C-terminus contains a unique, conserved MEEVD domain that
binds to tetratricopeptide (TPR) domain containing co-chaperones. The nucleotide
binding site lies in a deep pocket on the helical face of the N-domain. The adenine
base, sugar, and the α- phosphate group make extensive contacts within this pocket,
whereas the β- and the γ- phosphate groups display weak and no contacts respec-
tively. A hydrogen bond connects the adenine base with Asp79, while all other
contacts observed are polar in nature with water molecules interacting with the
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ribose sugar moiety. The α- and the β- phosphate groups are bound to an octahedrally
co-ordinated Mg2+ ion, making an indirect coupling with the protein(Pearl 2016;
Panaretou etal. 1998).
ATPase Cycle
HSP90s function as a chaperone is dependent on its inherent ATPase activity. The
molecule undergoes a series of conformational changes upon ATP and client protein
bindingthat culminatesin ATP hydrolysis and release of the partially folded client
protein(Li etal. 2012). Understanding the mechanism of this ATPase coupled con-
formational cycle has progressively advanced with several research groups using
sophisticated biophysical techniques like FRET and Analytical Ultracentrifugation
to identify the kinetic states in this cycle and the switch that occurs between these
states (Hessling etal. 2009; Mickler etal. 2009). The dening mechanism is the
‘molecular clamp mechanism’, (Ali et al. 2006) where ATP binding to the open
V-shaped constitutive HSP90 dimer (apo-state) induces structural changes via inter-
mediates, the formation of each of which is regulated by co-chaperones that have
specic roles to play in the cycle. The rate limiting step is the formation of a closed
‘tensed’ state, where the N-domains are dimerized and associated with the
M-domains. This closed conformation is committed to ATP hydrolysis withsubse-
quent release of the substrate and ADP occurring concomitantlyas the N-domains
dissociate and HSP90 returns to its open conformation, ready for another round of
theATPase cycle (Pearl 2016; Li etal. 2012). The rate limiting step involves major
structural changes occurring in the N-domain in two distinct ‘switch’ regions: (a)
the β-strand in the N-domain of one monomer hydrogen bonds with the main β-sheet
in the N-domain of the other monomer. Simultaneous movement of the α-helix
exposes a large hydrophobic patch that dimerizes with the equivalent patch of the
other monomer; and (b) the ‘lid’ segment, which ips over ~180° from its apo con-
formation to fold over the bound nucleotide in the pocket and ‘cradle’ the γ-phosphate
of ATP in a series of main-chain hydrogen bonds(Pearl 2016). Additionally, the
exible loop from the M-domain associates with the lid segment of the N-domain
via hydrophobic residues. This intra-molecular docking of the N and M-domains
facilitates the interaction between the γ-phosphate and theR380 residue, resulting
in the assembly of the two- halves of a split active site for ATP hydrolysis(Pearl
2016; Meyer etal. 2003a, b; Cunningham etal. 2012). The stable HSP90C-terminal
dimer model was challenged by Ratzke and his team (Ratzke et al. 2010) who
observed through FRET experiments that apart from the transient dimerization
observed at the N-domains, the C-terminus can also open and close with fast kinet-
ics, even when ATP/ADP is bound to the N-domain. They proposed a unique mech-
anism where HSP90 may undergo multiple transitions from being a dimer at the
C-domain and open at the N-domains, to being open at the C-terminus while
N-domains are dimerized, thereby associating a higher degree of dynamic exibility
that may inuence substrate release (Ratzke etal. 2010). The following schematic
depicts the conformational change from the open to the closed state of HSP90 via
several transition intermediates (Fig.8.3).
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HSP90 co-Chaperones
HSP90 can only keep itssubstrates in a folding competent state and prevent them
from undergoing aggregation, but cannot refold any of its clients by itself (Freeman
and Morimoto 1996). It requires the assistance of other proteins to carry out its
biological function. These proteins are also known as co-chaperones. Around 20
co-chaperones have been identied in eukaryotes and while some of them have been
well-characterized, the mechanism by which some of the other co-chaperones func-
tion is unclear. These co-chaperones mainly regulate the ATPase activity and bind-
ing of client proteins to HSP90 (Prodromou etal. 1999; 2002; Richter etal. 2004;
Roe etal. 2004; Chen and Smith 1998). They associate and dissociate dynamically
and help inthe transition from one intermediate conformation to another, or stabi-
lize a certain conformation in the protein folding cycle. Some of these co- chaperones
like HSP90/HSP70 Organizing Protein (HOP) (Johnson et al. 1998), protein
phosphatase PP5 (Silverstein et al. 1997) and PPIase family members FKBP51
Fig. 8.3 An overview of the conformational cycle of HSP90: Several co-chaperones and ATP
mediate the transition between the early, intermediate and late stages of substrate-bound HSP90
(Copyright clearance order number- 4177600710200)
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(Nair etal. 1997) and FKBP52 (Cox etal. 2007; Johnson and Toft 1994) have a TPR
domain that binds to the MEEVD sequence located in the C-terminus of the
chaperone (Scheuer etal. 2000; Das etal. 1998). Table8.2 (Modied from Li etal.
2012) lists some of the well-studied co-chaperones that have been identied and
their role in the ATPase cycle of Hsp90.
HSP90 co-chaperone Cycle
The most recent understanding of the chaperone cycle is that there are three differ-
ent complexes formed in a chronological fashion with different co-chaperone com-
position (Smith 1993). The rst complex, called the early complex consists of
HSP70, HSP40 and a client protein (Smith etal. 1992; Patricia Hernández etal.
2002; Cintron and Toft 2006), whichpresumably isa misfolded or nascent polypep-
tide. This complex docks to HSP90 via HOP which acts as a scaffold. One TPR
domain of HOP binds to one MEEVD motif of the HSP90 dimer while another
HOP domain binds to the early complex (Li etal. 2011). This HOP bound HSP90
complex can be called the intermediate complex. Addition of ATP and a PPIase
results in the formation of an asymmetric complex, where the TPR domain of the
PPIase binds with the unoccupied MEEVD motif of the other monomer, and
Table 8.2 List of some well-studied co-chaperones of HSP90 and their role in the ATPase cycle
(Modied from (Li etal. 2012))
Protein Name Function
TPR containing
co-chaperones
HOP Stabilizes the open conformation of HSP90; inhibits ATP hydrolysis;
simultaneously binds HSP90 and HSP70 and aids in transfer of nascent
polypeptides recognized by HSP70.
FKBP51/52 Maturation of steroid hormone receptors (SHR); chaperone.
CYP40 Maturation of Estrogen receptors specically; chaperone (Chen etal. 1998;
Pirkl and Buchner 2001).
PP5 Post translational modication of HSP90; dephosphorylates HSP90 and
CDC37; plays a role in processing of client proteins.
TPR2 Recognizes both HSP90 and HSP70 through its TPR domain; may act in the
client transfer from HSP70 to HSP90 (Brychzy etal. 2003).
Non-TPR
co-chaperones
AHA1 Stimulates ATPase activity; induces conformational change (Retzlaff etal.
2010; Sato etal. 2000).
P23 Binds and stabilizes closed client bound HSP90 heterocomplex; maturation
of client proteins; inhibits ATP hydrolysis; chaperone (Johnson and Toft
1994; Obermann etal. 1998; Bose etal. 1996; Freeman etal. 1996).
CDC37 Binds specically to client kinases and ‘presents’ them to HSP90 by binding
to HSP90s N-terminal domain via its C-terminal; inhibits ATP binding and
ATPase activity of HSP90; chaperone (MacLean and Picard 2003; Gaiser
etal. 2010; Siligardi etal. 2002; Ali etal. 2006).
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concomitantlyATP binds to the N-terminus (Smith 1993). The chaperone is still in
its open conrmation. HSP90 adopts the closed ‘committed to ATP hydrolysis’
conformation after p23 binds to the intermediate complex (Johnson et al. 1994;
Johnson and Toft 1995; McLaughlin etal. 2006; Freeman etal. 2000). This is called
the late complex where HOP and HSP70/40 dissociate from HSP90 as their binding
afnity is weakened due to the conformational change. After ATP hydrolysis, p23
and PPIase is released along with the partially folded client protein. Not much is
known about the ADP bound conformation that re-positions the relative orientations
of the N-domains just prior to the release of the client (Pearl 2016). Dynamic X-ray
scattering data has revealed that HSP90 can exist in a highly exible conformational
ensemble (Rice etal. 2008; Zhang etal. 2004), even in the nucleotide free state and
that the ADP bound state is a partially closed one with the N-domains close to each
other, but different to the ATP bound closed complex where the N-domains dimer-
ize (Scherrer etal. 1990).
Expression and Regulation of HSP90s Function under Thermal Stress
The upregulation of chaperones under heat shock conditions is mediated by the
Heat Shock Transcription Factor1 (HSF-1) (Fiorenza et al. 1995). HSF-1 under
normal conditions remains in its inactive monomeric state bound to HSP90. Upon
heat shock, HSP90 dissociates from the complex and HSF-1 undergoes trimeriza-
tion (Baler etal. 1993; Sarge etal. 1993). This trimer can bind to DNA regulatory
elementscalled the Heat Shock Elements (HSEs) and upregulate the transcription
of HSP genes like HSP90 and HSP70 (Akerfelt etal. 2010). Elevated levels of HSP
lead to inactivation of HSF-1; HSP90 and its cohorts FKBP2 and p23 bind tothe
trimeric state and attenuates its DNA binding afnity (Zou etal. 1998; Bharadwaj
etal. 1999; Guo etal. 2001) while HSP70/HSP40 bind to HSF-1 and prevent its
transactivation (Shi etal. 1998; Abravaya etal. 1992; Baler etal. 1992). This nega-
tive feedback loop regulates the chaperones at the transcription level, and thereby
modulates the levels of misfolded nascent polypeptides inside the cell. Additionally,
certain post translational modications affect the chaperone functions of HSP90
(Scroggins and Neckers 2007). Under normal circumstances, HSP90 remains exten-
sively phosphorylated, with as many as four phosphorylatedresidues in each iso-
form (Sefton etal. 1978; Kelley and Schlesinger 1982). Under heat shock conditions,
the general understanding is that HSP90 is rapidly dephosphorylated, leading to the
loss of HSP90s ability to stimulate the activity of its client proteins (Lees-Miller and
Anderson 1989; Morange and Bensaude 1991). Dephosphorylation is mediated by
Protein Phosphatase 1 (PP1), while phosphorylation is mediated by several kinases
like CKII, DNA-PK, and Akt (Wandinger etal. 2006; Dougherty etal. 1987; Walker
etal. 1985; Lees-Miller and Anderson 1989; Chalovich and Eisenberg 2012). After
clients that are bound to HSP90 under normal temperature get released due to
dephosphorylation, one of HSP90s client proteins, heme-regulated Inhibitor Kinase
(HRI), is activated by the chaperone upon rapid phosphorylation and this in-turn
down regulates protein synthesis by inactivating eukaryotic initiation Factor-2α
subunit (Scroggins and Neckers 2007). This cycle of dephosphorylation and phos-
phorylation of HSP90 reduces the load of misfolded proteins inside the cell.
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8.10 Combinatorial Assistance ofVarious Chaperones
There exists various checkpoints to ensure correct folding from the beginning of
protein synthesis tillit attains its native, biologically active conformation. Molecular
chaperones act as a crucial buffer providing conditions conducivefor the partially
unfolded intermediate to fold. The chaperone machinery helps newly synthesized
protein to navigate the complex energy landscape in order to achieve their native
form. The following paragraph describes the presence of various chaperones at
different spatial and temporal points, coordinating with each other to regulate
protein folding.
The chaperones are present at different levels: The rst chaperonic tier consists
of ribosome associated chaperones. These chaperones stabilize the nascent poly-
peptides which are being synthesized on the ribosome and initiate their folding
process. The second tier of components act subsequently and aid in the complete
folding of the proteins. Both systems cooperate to form one single folding pathway.
Chaperones involved in the rst tier include prokaryotic chaperones Trigger Factor
(TF) and eukaryotic Ribosome Associated Complex (RAC) (Kramer et al. 2009).
These are present on the large ribosome near the exit tunnel of a polypeptide chain.
This RACcomplex comprises HSP70 homologs Ssb1, Ssb2 and Ssz1, zuotin (in
yeast) and the corresponding homologs in higher eukaryotes (Hundley etal. 2005;
Otto et al. 2005). These chaperones primarily bind to the exposed hydrophobic
regions of the proteins. TF works in an ATP independent manner to fold a newly
synthesized polypeptide. The bound polypeptide tries to bury its hydrophobic
regions which facilitate the foldingprocess. Interestingly most of the nascent chains
(~70%) seek the assistance of TF and are successfully folded without any further
assistance. Small proteins also fold spontaneously after their synthesis without any
further assistance (Ferbitz etal. 2004; Merz etal. 2008).
Another class of proteins (~ 20%) possess strong hydrophobic elements (Teter
etal. 1999; Thulasiraman et al. 1999) and thus TF and other ribosome associated
chaperones do not provide enough assistance for their folding. It has been reported
that TF dissociates from the ribosome while bound to the polypeptide chain (Agashe
etal. 2004), thereby presenting the unfolded polypeptides to the downstream chap-
erones like DnaJ/DnaK (Martinez-Hackert and Hendrickson 2009). The DnaJ/
DnaK system further interacts with the longer polypeptide chains and helps in their
folding in an ATP dependent manner. In eukaryotes the second tier also consists of
NAC (Nascent chain associated complex) and like TF, it interacts with the newly
synthesized polypeptides and helps them attain their folded conformation with the
assistance of RAC, HSP70, and other cofactors.
HSP70s along with other cofactors like HSP40, J-proteins and various NEFs aid
in the folding of substrates in an ATP dependent manner (Zhu etal. 1996; Mayer
etal. 2000; Pellecchia etal. 2000). HSP40 also binds with the misfolded polypep-
tides and recruits HSP70 to assist in the proper folding of substrates (Young etal.
2003). In eukaryotes, subsequent to the HSP70 system, HSP90 with the help of
numerous regulators and co-chaperones acts as a nisher, helping the polypeptide
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attain its biologically active structure (Pearl and Prodromou 2006; Zhao and Houry
2007; Scheuer etal. 2000). The remaining 10% of substrates that remain in a non-
functional, yet non-aggregated state, are shifted to the chaperonin cage for their
folding (Hartl 1996; Horwich etal. 2007). The environment in the nano cage of
chaperonin facilitates the misfolded proteins to attain a native-like structure
(Gromiha and Selvaraj 2004). In bacteria, GroEL/ES help in thefolding of substrate
proteins. In eukaryotes, substrate proteins are presented to the chaperonin TRiC.The
reaction is mediated by HSP70 and Prefoldin which interact directly with the TRiC
system resulting in the release of folded, functional proteins (Hartl and Hayer-Hartl
2002).
8.11 Conclusions
Compilation of studies both past and recent, on chaperone mediated protein folding
unequivocally show that chaperones play a basal role in maintaining protein homeo-
stasis inside the cell. While some of them function as ‘foldases’, hydrolysing ATP
to carry out the folding and release of various substrates, a few others function
independent of ATP, primarily as ‘holdases’. Under stress conditions however,
chaperones undergo multiple levels of modication; from the enhanced rates of ATP
hydrolysis andmore efcient substrate binding to various post-translational modi-
cations that aid in the transcriptional upregulation of several hsp genes. Chaperones
can also prevent aggregation of misfolded proteins that tend to accumulate under
heat shock conditions. Chaperones play a dual role inside the cell; either rescuing
misfolded or unfolded polypeptides and preventing aggregation or triggering degra-
dation of substrates when cell damage is irreversible, both of which needs to be
tightly regulated to maintain proteostasis. Such properties of chaperones are cur-
rently being utilized in the industry as well as in designing therapy for certain debil-
itating diseases like cancer and neurodegenerative disorders. Overexpression of
certain proteins can be a major problem, especially when they are being expressed
in a different host. Several studies including some of our own have shown that
achaperone or a combination of chaperones have been proven effective in increas-
ing the yield as well as the active fraction of total protein expressed. Some of those
proteins are considered therapeutically important; hence, large-scale productions of
such proteins are regularly uptaken by the pharmaceutical industry. Chaperones
thus ablate a signicant clog in this giant wheel of industrial-scale protein produc-
tion and such strategies have come to the rescue over the years. HSP90 has been
used as a target to design inhibitors that have been successful in the amelioration of
tumor progression and development, and is considered a hot prospect for drug-
based therapy for the treatment of certain types of cancer. Although a promising
approach, only a few compounds have reached the clinical trials and none of them
have made it to the market. On the other end of the spectrum, the ability to prevent
aggregation of misfolded proteins and even refold partially folded ‘aggregation-
prone’ intermediates can be used to treat neurodegenerative disorders. We are
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currently working on one such compound that stimulates the chaperone functions of
HSP90 in-vitro, and plan to carry out several studies to investigate its potential in
preventing certain neurodegenerative conditions. Overall, chaperones make an
interesting topic of study, not only because they play such important roles in regu-
lating protein function, stability and degradation but also because they possess tre-
mendous value both industrially and therapeutically.
Acknowledgments The authors acknowledge the nancial assistance from IIT Delhi and infra-
structural facility from IIT Delhi, India. AS and AP acknowledge nancial assistance from CSIR,
Government of India for providing fellowships in their doctoral course programme. SP acknowl-
edge nancial assistance from UGC, Government of India for providing fellowships in their doc-
toral course programme. BKC acknowledges IIT Delhi for providing fellowship in the doctoral
course program.
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