AN INTRODUCTION TO

MOLECULAR CHAPERONE
WTREN
4/12/2012

Definition of Chaperone
A large group of unrelated protein families whose role is to
stabilize unfolded proteins, unfold them for translocation
across membranes or for degradation, and/ or to assist in their
correct folding and assembly.
Proteins that assist the non-covalent folding or unfolding and
the assembly or disassembly of other macromolecular
structures, but do not occur in these structures when the
structures are performing their normal biological functions
having completed the processes of folding and/or assembly.
From Wikipedia
Any proteins that interacts with, stabilizes or helps another
protein to acquire its functionally active conformation, without
being present in its final structure.
From M H-Hartl

Properties of Chaperone
Molecular chaperones interact with unfolded or partially
folded protein subunits.
They stabilize non-native conformation and facilitate correct
folding of protein subunits.
They do not interact with native proteins, nor do they form
part of the final folded structures.
Some chaperones are non -specific, and interact with a wide
variety of polypeptide chains, but others are restricted to
specific targets.
They often couple ATP binding/hydrolysis to the folding
process.
Essential for viability, their expression is often increased by
cellular stress.

Classification of Chaperone
"Folding" chaperones (e.g., DnaK and GroEL):
rely on ATP-driven conformational changes to mediate the net
refolding/unfolding of their substrates.

"Holding" chaperones (e.g., Hsp33, Hsp31 and IbpB):

maintain partially folded proteins on their surface to await
availability of "folding" chaperones upon stress abatement.

The third class of chaperone (e.g., ClpB):

promotes the solubilization of proteins that have become aggregated
as a result of stress

Examples Of Chaperone

Classification Of The Human Heat Shock

Proteins
HSPH (HSP110), HSPC (HSP90), HSPA (HSP70), HSPD/E
(HSP60/HSP10), CCT (TRiC)
DnaJ (HSP40)
HSPB (small HSP or sHSP)
Some proteins are ATP dependent, and they are involved in
co-translational or posttranslational protein folding.
Other proteins (such as DNAJ) play the role of co-chaperones
modulating activity of the other heat shock proteins.
The small heat shock proteins (HSPB) are responsible for the
binding of improperly folded protein -substrates and their
further transfer to the ATP -dependent chaperones or to the
protein degradation machines like proteasomes or
autophagosomes .

HSP70
Heat shock 70kDa proteins(HSP70s)
Ubiquitous molecular chaperones that function in a myriad
biological processes:
Modulating polypeptide folding
Degradation and translocation across membranes
Protein-protein interactions

HSP70s have high sequence identity and share a single

biochemical activity:
An ATP-dependent client-binding
Release cycle combined with client protein recognition

Structures Of HSP70
I t i s c o m p os ed o f a C - t e r mi n a l
s u b s t r a t e - b i n d i n g d o m a i n ( S B D) a n d
a N - t e r mi n a l n u c l e o t i d e - b i n d i n g
domain(NBD or ATPase domain).
T h e t w o d o m a i n s a r e c o n n e c te d b y a
short, flexible, hydrophobic linker.
The SBD is composed of a 25kDa sandwich subdomain and a 10kDa
helical lid domain.
T h e N B D c o n s i s ts o f t w o l o b e s w i t h
a d e e p c l e f t b e t w een t h e m, a t t h e
bottom of which nucleotide binds.
The -sandwich of the SBD
recognized extended, ~7 -reisue
s e g me n t s e n r i c h e d i n h y d r o p h o b i c
amino acids, preferentially when
t h e y a r e f r a m ed b y p o s i t i v e l y
charged residues.

The Core HSP70 Machinery

HSP70s invariably require a J protein(HSP40 or DnaJ) and,
almost always, a nucleotide exchange factor (NEF) as partners.
The HSP70 cycle:
HSP70 cycles between ATP- and ADP-bound states.
On and off rates for client binding are rapid in the ATP -bound state
and slow in the ADP-bound state.
ATPase activity is stimulated by J proteins, facilitating client capture;
dissociation of ADP is stimulated by NEFs.

Dynamics Of HSP70
The -helical lid and a
conformational change in the
-sandwich domain regulate
the affinity state for the
peptide in an ATP -dependent
manner:
In the ATP bound state, the lid
adopts an open conformation,
resulting in high on and off rates
for peptide;
Hydrolysis of ATP to ADP is
strongly accelerated by HSP40,
leading to lid closure and stable
peptide binding.
After ATP hydrolysis, a
nucleotide-exchange factor binds
to the HSP70 ATPase domain and
catalyzes ADP-ATP exchange,
resulting in lid opening and
substrate release.

J Proteins Drive The Multi-functionality

Of HSP70S
The number of HSP70s in each cell is rather limited. However,
J proteins often far outnumber HSP70s and NEFs in a cellular
compartment.
J proteins contain a highly conserved ~70 amino acid
signature region, the J domain.

J Protein Function

HSP90
Hsp90 can bind and hydrolyze ATP and is believed to interact
with hydrophobic surfaces on substrate (client) proteins.
In all eukaryotic cells their main function is believed to be the
interaction with a defined set of proteins, called clients, in a
native or near-native state. It forms a proteostasis hub that
controls numerous important signaling pathways in eukaryotic
cells.
Particular Hsp90 clients, such as p53, Cdk4, c -Src/v-Src and
HER-2/HER-3, are oncogenic or otherwise required for cell
proliferation, making Hsp90 an attractive drug target.
Hsp90 has been shown to be a highly flexible and dynamic
molecule, and large conformational changes are coupled to
ATP binding and hydrolysis .
Hsp90 is highly conserved from bacteria to eukaryotes with
~50% sequence similarity between E coli and humans.

Architecture Of HSP90
HSP90 functions as a dimer of subunits.
Hsp90 proteins consist of three functional domains:
The N-terminal NBD, which binds and hydrolyses ATP.
The middle domain (MD), which is involved in client and co-chaperone
binding.
A C-terminal dimerization domain (DD).

 Hsp90 proteins have a large

degree of conformational
freedom.
HSP90 dimer undergoes an
ATP-driven reaction cycle :
ATP binding leads to the
dimerization of the N-terminal
domains, forming the HSP90
'molecular clamp'. This results
in a compaction of the HSP90
dimer.
After hydrolysis, the ATPase
domains dissociate, and the
HSP90 monomers separate Nterminally.

 The flexibility of Hsp90 is functionally important , and that

dif ferences in conformational flexibility among Hsp90
homologs may relate to dif ferences in how they interact with
clients.
The conformational equilibrium of Hsp90 plays a central role
in client interactions, and is possibly tuned to the client
requirements of each organism.
Various cofactors regulate HSP90s ATPase cycle:
Cdc37, p23/Sba1, Aha1

These factors are thought to adjust the kinetic properties of

the cycle to achieve certain conformational transitions in
HSP90-bound substrates, as well as their release from HSP90.

Small HSP(Small Heat Shock Protein)

Expressed throughout nearly all kingdoms
(except for certain bacteria species).
Usually with monomer molecular mass in the
range of 1243 kDa.
All proteins of this family contain the so called -crystallin domain(C domain or ACD),
a region of 90 residues. This domain is
considered as an important hallmark of small
heat shock protein, independent of their origin
and nature.
The -crystallin domain consists of six to
eight -strands organized in two -sheets
f l a n k e d b y t h e N H 2- a n d C O O H - t e r m i n a l
regions, which are variable in length and
structure.
sHSP shows extensive sequence variation and
evolutionary divergence .
Many sHSPs have been shown to act in an
ATP-independent fashion to bind up to an
equal weight of non-native protein to limit
aggregation and to facilitate subsequent
refolding by ATP -dependent chaperones

Dimer Structures
The majority of sHSPs exist as
oligomers of between 12 to >48
subunits in their native state.
In all sHSP oligomers , a dimer is
the basic building block.
Proteins containing the 6 loop
form dimers through strand
swapping. Dimers of this type
are predicted for most bacterial,
yeast and plant sHSP.
In the absence of 6, 7 is
elongated and forms the dimer
interface in anti-parallel
orientation with 7 of the other
monomer.

Oligomeric Architectures
The C-terminal I/LXI/L
motif, patches a
hydrophobic groove formed
by 4 and 8 on one edge
of the ACD -sandwich.
Some contacts distal to the
I/LXI/L motif may be more
important for assembly of
these oligomers.
Perhaps the extended
length of the N-terminal
arm allows other ways of
oligomeric assembly.

The Chaperone Mechanism Of sHSP

Other HSPs
HSP100:
The Hsp100/Clp family of chaperones belongs to the superfamily of
AAA+ domain-containing ATPases associated with various
cellular activities.
Hsp100 proteins contain one or two AAA+ domains arranged in
hexameric rings with a central pore through which substrate proteins can
be threaded. Many Hsp100 proteins (ClpA, ClpX, HslU in E. coli) associate
with ring-forming peptidases (ClpP, HslV), unfold proteins, and feed them
into the proteolytic chamber.

HSP33:
A redox-regulated chaperone that stabilizes proteins unfolded by severe
oxidative stress.

HSP31:
A heat-inducible homodimeric chaperone
Its functions as a holding chaperone that helps cells handle protein
unfolding under extremes of temperature and plays a central role in the
ability of starved E. coli to survive acid stress.

Main References
Saibil, H. R. (2008). "Chaperone machines in action." Current
Opinion in Structural Biology 18(1): 35-42.
Mayer, M. P. (2010). "Gymnastics of Molecular Chaperones."
Molecular cell 39(3): 321-331 .
Mymrikov, E. V., A. S. Seit-Nebi, et al. (2011). "Large Potentials
of Small Heat Shock Proteins." Physiological Reviews 91(4):
1123-1159.
Hartl, F. U., A. Bracher, et al. (2011). "Molecular chaperones in
protein folding and proteostasis." Nature 475(7356): 324-332.
Basha, E., H. ONeill, et al. (2012). "Small heat shock proteins
and -crystallins: dynamic proteins with flexible functions."
Trends in Biochemical Sciences 37(3): 106-117.
Http://faculty.washington.edu/baneyx/Chaperones/Chaperones.
html
Http://people.cryst.bbk .ac.uk/~ubcg16z/hsplec.html

THANKS