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CHAPERONES

MIREN AIZPIRI
KANISHKA BHAMBI
JAIME CANO

INDEX
INTRODUCTION

HSP 60 (GroEL)
HSP 70
HSP 90
CONCLUSIONS

CHAPERONES
Definition: An abundant class of proteins that
assist in the correct folding and maturation of
other cellular proteins.
Dont participate in the final
mature structure.
Have little specificity for their
substrates.
Rarely function alone.
Hartl FU, et al. Molecular chaperones in protein folding and proteostasis. Nature.
Nature Publishing Group; 2011 Jul 21 ;475(7356):32432.

HEAT SHOCK PROTEINS


Are found in all living organisms, from bacteria to humans.
Induced by stress they participate in:
Refolding of stress-denatured polypeptides
Prevent polypeptide aggregation.

They also work under non-stress conditions.


2 types:
Intracellular: Protective function.
Extracellular and membrane bound: Immunological function.

HEAT SHOCK PROTEINS


ATP DEPENDENT
Mammals

Prokariotes

HSP 60

GROEL

HSP 70

DnaK

HSP 90

HptG

HSP 100

Clp
ATP INDEPENDENT
Small HSPs

HSP 60
HSP60

INTRODUCTION
Highly conserved
Present in mythocondria and
citoplasm
Protein folding and transport
DNA metabolism
Apoptosis

GroEL
PDB: 1AON

First large oligomeric chaperone (1970s)


Essential for cell survival
50% of proteins interact with it
30% are unable to fold whithout it

GroES

GroEL

STRUCTURE

14 identical subunits arranged in two heptameric


rings stacked back-to-back
GroES

Each subunit
Apical domain
GroEL

Intermediate domain
Ecuatorial domain
PDB: 1AON

GroEL

APICAL DOMAIN

Polypeptide binding site


GroES binding site

Root: Scop
Fold: Alpha and beta proteins (a/b)
Class: The "swivelling" beta/beta/alpha
domain
Superfamily: GroEL apical domain-like
PDB: 1AON

GroEL

INTERMEDIATE DOMAIN

Articulated Joint between


Apical and Equatorial
domains

Root: Scop
Fold: Alpha and beta proteins (a+b)
Class: GroEL-intermediate domain like
Superfamily: GroEL-intermediate
domain like
PDB: 1AON

GroEL

ECUATORIAL DOMAIN

ATP binding site


Inter and intra ring contacts

Root: Scop
Fold: GroEL equatorial domain-like
Class: All alpha proteins
Superfamily: Equatorial domain-like
PDB: 1AON

GroEL

MECHANISM OF ACTION

Clare DK, et al. ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the GroEL chaperonin. Cell. 2012
Mar 30;149(1):11323.

GroEL

PROTEIN BINDING SITE

Buckle AM et al. A structural model for GroEL-polypeptide


recognition. Proc Natl Acad Sci U S A . 1997 Apr;94(8):35715.

PDB: 1AON

GroEL

PROTEIN BINDING SITE

Groel T state side view

PDB: 1OEL

GroEL

Y199
S201
Y203
F204

PROTEIN BINDING SITE

GroEL
L234
L237
L259
V263
V264

PROTEIN BINDING SITE

GroEL

PROTEIN ENCAPSULATION

ATP

PDB: 1CXK

T State

PDB: 4AAQ

RS1 State

PDB: 1AON

RS2 State

PDB: 4AAS

RS-open State

PDB: 2C7D

R-ES State

GroEL

PROTEIN ENCAPSULATION

PDB white: 1CXK / PDB yellow: 4A


Left side view

Frontal view

Right side view

E255-K207

R197-E386

PDB: 1CXK

E255-K452

K80 -E386

PDB: 4AAQ

PDB: 4AAS

K80 -E386

PDB: C2D7

GroEL

K80

R197
K207

PROTEIN ENCAPSULATION

GroEL
K245

E386

PROTEIN ENCAPSULATION

GroEL
R452

PROTEIN ENCAPSULATION

GroEL

PDB: 1AON

ATP BINDING POCKET

D398A

D398

GroEL
T30
L31
G32
P33
K51
D52
G53

D87
G88
T89
T90
T91
I150
S151

ATP BINDING POCKET

GroEL
D398
G414
G415
G416

I454
I493
D495

ATP BINDING POCKET

GroEL

HYDROPHYLIC SURFACE

Hydrophylic inner ring surface

PDB: 1AON

GroEL
K4

K42
E61
K75
D85

D196
R197

HYDROPHYLIC SURFACE

GroEL
K266
E252
D253
E255
E257
K277
D283
R285
K286
K327
D328
D359
E363
K364
E367
R368
K371

K380
E386
K393

HYDROPHYLIC SURFACE

GroEL
R404
E408

D523

HYDROPHYLIC SURFACE

GroEL

STAMP

PDB red: 1AON / PDB blue: 4V40 / PDB white: 1IOKA / PDB yellow: 1SJPA

Cluster: 1 ( 1AONA & 4V40a ) Sc 8.43 RMS 1.41


Cluster: 2 ( 1IOKA & 1SJPA ) Sc 5.71 RMS 1.88
Cluster: 3 ( 1AONA 4V40a & 1IOKA 1SJPA ) Sc 5.92 RMS 2.68

GroEL

STAMP

PDB red: 1OEL / PDB blue: 4V40 / PDB white: 1IOKA / PDB yellow: 1SJPA

Cluster: 1 ( 1oelA & 1IOKA ) Sc 8.20 RMS 1.16


Cluster: 2 ( 1SJPA & 1oelA 1IOKA ) Sc 7.86 RMS 2.11
Cluster: 3 ( 4V40A & 1SJPA 1oelA 1IOKA ) Sc 5.80 RMS 2.53

HSP 70

INTRODUCTION

Evolutionarily conserved group of molecular chaperones.


Found in all kingdoms from archaebacteria to humans.
While most prokaryotes have only one Hsp70 gene, some gramnegative bacteria an all eukaryotes encode several Hsp70
proteins.
Considered a cancer-critical survival protein. It is
constitutively overexpressed in most human cancer cells

HSP70 is not essential for viability.

HSP 70

EVOLUTION

Two large divisions:


o prokaryotic DnaK proteins together
with the plastidal and mitochondrial
sequences

Proof for the


endosymbiont
theory

o eukaryotic nucleo/cytoplasmatic
and ER proteins

Possible explanation: there


have possibly been (at least)
two ancestral genes, of which
one got lost in different
lineages
Rensing, S. a. (1994). Journal of Molecular Evolution,
8086.

HSP 70

STRUCTURE

N-terminal nucleotide binding domain (NDB)


ATPase activity
C-terminal substrate binding domain (SBD)
NRLLLTG interacts with high affinity
If the NBD is bound to ADP
However, when ATP is bound to the NBD
Substrate binds significantly more weakly

Bukau, B., & Horwich, A. L. (1998). The Hsp70 and Hsp60 chaperone machines. Cell,
92, 351366. doi:10.1016/S0092-8674(00)80928-9

HSP 70

STRUCTURE

Highly conserved DVLLLD-Linker segment of DnaK


Thr13, Thr14, and Asp366 likely candidates for transmitting the
information about catalytic events in the active site to the SBD.

Qi, R., Sarbeng, E. B., Liu, Q., Le, K. Q., Xu, X., Xu, H., Liu, Q. (2013). Allosteric opening of the polypeptide-binding site
when an Hsp70 binds ATP. Nature Structural & Molecular Biology, 20(7), 900907.

HSP 70

N-TERMINAL DOMAIN

44kD, 388 amino acid.


NBD consists of two large,
globular subdomains (I
and II), each further
divided into two small
subdomains (A and B)
separated by a deep
central cleft.
DnaK (bacterial Hsp70)
has been extensively
studied at the structural
level.

IB

IIB

IA
IIA

PDB:1S3X

HSP 70

Human Hsp70 - NBD

PDB:1S3X

PDB:1SX3

HSP 70

SEQUENCE ALIGNMENT

PDB:1S3X

Highly conserved
amino acids: T13,
K71, and T204

PDB:1SX3

HSP 70

Human Hsp70 - NBD


PDB:1S3X

New metal binding


motif at the
junction between:
- a beta sheet (190225)
- and alpha helix (230250)
- Close to the
catalytic site

PDB:1SX3

HSP 70

C-TERMINAL DOMAIN

Is responsible for the substrate binding (SBD).


Composed of a two-layered -sandwich (SBD), which contains the
peptide binding pocket, and an -helical subdomain (SBD).

PDB:4PO2

HSP 70

DnaK - SBD

Root: scop
Class: All beta proteins
Fold: Heat shock protein 70kD
(HSP70), peptide-binding
domain
beta-sandwich: 8 strands
in 2 sheets

PDB:4EZW

HSP 70

HYDROPHOBIC CONTACTS
PDB:4EZW

Residues of the base:


Ile401, Thr403,
Met404, Val407,
Thr409, Ala429,
Gln433, Ala435,
Val436, Ile438,
Ile472.
Hydrophobic core
Surface potential
negatively charged
but neutralized by
basic segments

HSP 70

SUBSTRATE BINDING GROOVE

Structural alignment
Superimposition between DnaK
and the human Hsp70

PDB:1DKZ,blue

Sc = 3.75
RMSD = 1.61

Existence of considerable
structural variability within the
SBD region

PDB:4PO2,magenta

HSP 70
Structural alignment Alpha
domains of the SBD of Rat
Hsc70 and our Hsp70
Sc = 0.51
RMSD = 2.23

SBD subdomain is
inherently more flexible than
the SBD confirmed after
using ALIGNFIT

SBD REGION
PDB:4PO2

PDB:1UD0

HSP 70

ALIGNFIT

HSP 70

HSP70 ISOFORMS

Structural alignment
between 4 different
isoforms of human Hsp70
(3FE1, 3GDQ, 3I33, 3JXU)
Constitutively expressed
Hsp70 housekeeping
functions
Stress-induced Hsp70
by heat stress, heavy
metals, ischemia, etc.
Sc = 5.80
RMSD = 0.48

PDB: 3FE1, 3GDQ, 3I33,3JXU

HSP 90

INTRODUCTION

Comprises 1-2% of total cellular protein content.


In vertebrates:

HSP 90 : inducible
ESSENTIAL for the viability of the cell
HSP 90 : constitutive
Grp94: in the endoplasmic reticulum
TRAP1: in the mitochondria

Principal clients:
Nuclear hormone receptors
Protein kinases

HSP 90

EVOLUTION

Its found in bacteria and all branches of


eukarya, but it is apparently absent in archaea.

Gene duplications:
Cytosolic and ER Paralogous
Alpha and Beta isoforms
Yeast constitutive and heat-inducible forms

Mitochondrial TRAP1 origin is unclear.

Gupta RS. Phylogenetic Analysis of the 90 kD Heat Shopck Family of Protein Sequences and an Examination
of the Relationship among Animals, Plants, and Fungi Species. Mol. Biol. Evol. 1995;12(6):1063-1073.

HSP 90

STRUCTURE

Globular protein, non-polar on the inside and polar on the outside.


Forms a homodimer, which each subunit comprising 3 domains.
Aa number:
Domains:

Function:

210

272

629

N-terminal

CR

Middle

N-terminal

CR

Middle

ATP binding

Client binding

732

Dimerization
ATP binding

HSP 90

MECHANISM OF ACTION
ATP binding

2IOQ

2O1V
OPEN

2CG9
CLOSE

ATP hydrolysis = ADP + Pi

HSP 90

N-TERMINAL DOMAIN

Root: Scop
Class: Alpha + Beta
Fold: ATPase domain of HSP90 chaperone/DNA topoisomerase II/histidine kinase
PDB: 1AM1

Eigh-stranded
antiparallel beta sheet
covered on one face by
nine alpha helices, four
of them are 310 type.

HSP 90

N-TERMINAL DOMAIN

E. Coli DNA Gyrase B and yeast HSP90 superimposition.


They have a common
ancestor, but diverged
early in evolution.
Score: 3.85
RMS: 2.13

1AJ6

1AM1

HSP 90

N-TERMINAL DOMAIN

E. Coli DNA Gyrase B and yeast HSP90 sequence alignment.

HSP 90

N-TERMINAL DOMAIN

Human and yeast superimposition

Score: 9.11
RMS: 0.67

1YET

1A4H

HSP 90

MECHANISM OF ACTION
H5

N-terminal domain

H5
H9
L2
H2

L2 and H5 form
the lid
that
L2
constricts the
pocket entrance.
H4

L1

H4

H2

1AM1

HSP 90

MECHANISM OF ACTION
H5

Superimposition
H9
of the N-terminal
domain in open
and close
L2
conformation
H4

L1

H5

L2

H2

1YES

1YER

HSP 90

N-TERMINAL ATP POCKET


15 A deep.
A half of the 17 aa lining its
interior are hydrophobic,
a quarter polar, and a
quarter charged.

PDB: 1AM1

HSP 90

N-TERMINAL ATP POCKET

2CG9

HSP 90

MUTATIONS

ATP binding and hydrolysis are required for HSP90 function in vivo.
D79N yeast mutant bound ATP much less efficiently than the WT.
E33D hydrolyzed ATP with similar Kcat values, although the Km value is higher
than the WT.
E33A doesnt have the ability to hydrolyze ATP.
T101I stabilizes the open conformation and decreases ATPase activity.
A107N stabilizes the closed conformation through the formation of additional
HBs and increases ATPase activity.

HSP 90

CLUSTAL N-TERMINAL
Glu: ATP hydrolysis
Asp, Asn: ATP binding
GxxGxG motif of
the lid.

HSP 90

CLUSTAL N-TERMINAL

Different HSP90 isoforms sequence alignment

Glu: ATP hydrolysis

Asp, Asn: ATP binding

GxxGxG motif of the lid

HSP 90

MIDDLE DOMAIN

Root: Scop
Class: Alpha + Beta
Fold: Ribosomal protein S5 domain 2-like
PDB: 3PRY

sandwich

sandwich

3 short helices arranged


in a right-handed coil

HSP 90

MIDDLE DOMAIN

Human and yeast superimposition


3PRY

Score: 7.47
RMS: 1.67

1HK7

HSP 90

CLUSTAL MIDDLE DOMAIN


Trp-300
Arg-380

2CG9

HSP 90

CLUSTAL MIDDLE DOMAIN

Varible region rich in acidic aa: Shielding the DNA-binding domain of steroid
hormone receptors.

HSP 90

C-TERMINAL DOMAIN

Root: Scop
Class: Alpha + Beta
Fold: HSP90 C-terminal domain
PDB: 2CG9

A short helix (H1) leads


to a small 3-stranded
antiparallel sheet.

H2

H4
H1

H5

The loop between strands


b and c contains a second
short helix (H2).
H3, H4 and H5.

H3

HSP 90

CLUSTAL C-TERMINAL

ANMERIMKA: binding of
glucocorticoid receptor.
(M)EEVD motif: binding of TPR-domain
containing co-chaperones.

HSP 90

(M)EEVD MOTIF

Recruits TPR-domain containing co-chaperones.


Consists of a 34 aa helix-turn-helix motif, which forms a superhelical groove
that interacts with the (M)EEVD motif.
1ELR

HSP 90
1ELR

(M)EEVD MOTIF

(M)EEVD MOTIF
This motif is also conserved in the HSP 70
C-terminal domain.

HSP 90

CANCER

Increased expression of HSPs above the level observed in normal tissues.


HSP 90 allows mutant proteins to retain or even gain function and thus
promotes tumor cells survival and proliferation.
A good target for cancer therapy would be the inhibition of Hsp90.
Geldanamycin
Benzoquinone

Carbamat group
Ansa ring

Inhibition of ATP binding and


hydrolysis

Degradation of oncogenic
HSP 90 clients by proteasome

HSP 90

GELDANAMYCIN

Superimposition of Geldanamycin-bound N-terminal


domain with ATP-bound N-terminal domain.
ATP in red
Geldanamycin in blue

1YET

1AM1

Score: 9.06
RMSD: 0.71

HSP 90
5 HB:
- with Lys-112
- with Lys -58

- with Asp-93
- with Thr-184
- with Phe-138

GELDANAMYCIN

CONCLUSIONS
HSPs are the most conserved proteins present in
both prokaryotes and eukaryotes.
HSP60: we expect the structure to be rather conserved.
HSP70: the NBD and the SBD are conserved, although
the alpha region of SBD is quite variable.
HSP90: the most conserved domain is the N-terminal
domain.

PDB Ids
PDB ID

DESCRIPTION

1S3X

The crystal structure of the human Hsp70 ATPase domain

4PO2

Crystal Structure of the Stress-Inducible Human Heat Shock Protein


HSP70 Substrate-Binding Domain in Complex with Peptide Substrate

4EZW

Crystal structure of the substrate binding domain of E.coli DnaK in


complex with the designer peptide NRLLLTG

1DKZ
1UD0

The substrate binding domain of dnak in complex with a substrate


peptide, determined from type 1 native crystals
CRYSTAL STRUCTURE OF THE C-TERMINAL 10-kDA SUBDOMAIN OF
HSC70

3FE1

Crystal structure of the human 70kDa heat shock protein 6 (Hsp70B')


ATPase domain in complex with ADP and inorganic phosphate

3GDQ

Crystal structure of the human 70kDa heat shock protein 1-like


ATPase domain in complex with ADP and inorganic phosphate

3I33
3JXU

Crystal structure of the human 70kDa heat shock protein 2 (Hsp70-2)


ATPase domain in complex with ADP and inorganic phosphate
Crystal structure of the human 70kDa heat shock protein 1A (Hsp701) ATPase domain in complex with ADP and inorganic phosphate

PDB Ids
PDB ID

DESCRIPTION

1AM1

ATP BINDING SITE IN THE HSP90 MOLECULAR CHAPERONE

1AJ6

NOVOBIOCIN-RESISTANT MUTANT (R136H) OF THE N-TERMINAL 24


KDA FRAGMENT OF DNA GYRASE B COMPLEXED WITH NOVOBIOCIN
AT 2.3 ANGSTROMS RESOLUTION

1YET

GELDANAMYCIN BOUND TO THE HSP90 GELDANAMYCIN-BINDING


DOMAIN

1A4H

STRUCTURE OF THE N-TERMINAL DOMAIN OF THE YEAST HSP90


CHAPERONE IN COMPLEX WITH GELDANAMYCIN

3PRY

Crystal structure of the middle domain of human HSP90-beta refined


at 2.3 A resolution

1HK7

MIDDLE DOMAIN OF HSP90

2CG9

CRYSTAL STRUCTURE OF AN HSP90-SBA1 CLOSED CHAPERONE


COMPLEX

2O1V

Structure of full length GRP94 with ADP bound

1YER

HUMAN HSP90 GELDANAMYCIN-BINDING DOMAIN, "CLOSED"


CONFORMATION

PDB Ids
PDB ID

DESCRIPTION

1YES

HUMAN HSP90 GELDANAMYCIN-BINDING DOMAIN,


"OPEN" CONFORMATION

1HJO

ATPase domain of human heat shock 70kDa protein 1

1AON

CRYSTAL STRUCTURE OF THE ASYMMETRIC


CHAPERONIN COMPLEX GROEL/GROES/(ADP)7

1XCK

Crystal structure of apo GroE

4AAS

ATP-triggered molecular mechanics of the chaperonin GroEL

4AAQ

ATP-triggered molecular mechanics of the chaperonin GroEL

2C7D

Fitted coordinates for GroEL-ADP7-GroES Cryo-EM complex


(EMD-1181

1IOK

CRYSTAL STRUCTURE OF CHAPERONIN-60 FROM


PARACOCCUS DENITRIFICANS

4V40

Crystal Structure of the Chaperonin Complex


Cpn60/Cpn10/(ADP)7 from Thermus Thermophilus

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Saibil HR, Fenton W a., Clare DK, Horwich AL. Structure and allostery of the chaperonin GroEL. J Mol Biol [Internet]. Elsevier Ltd; 2013;425(9):147687. Available from:
http://dx.doi.org/10.1016/j.jmb.2012.11.028
Xu Z, Horwich a L, Sigler PB. The crystal structure of the asymmetric GroEL-GroES-(ADP)7 chaperonin complex. Nature. 1997;388(1994):74150.
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9];475(7356):32432. Available from: http://www.nature.com.sare.upf.edu/nature/journal/v475/n7356/full/nature10317.html

PEMS
1) The Heat Shock Protein 90:
a) Has 3 domains: N-terminal, middle,
and C-terminal
b) Requires Calcium for its function
c) Is absent in non-stress conditions
d) Is not conserved among different
species
e) Is an essential protein in prokariotes

4) The nucleotide binding domain (NBD) of the Hsp70 chaperon


family:
a) Has ATPase activity
b) Consists of three globular domains
c) When is bound to ADP interacts more weakly with the substrate
d) When is bound to ATP interacts with high affinity with the
substrate
e) Always interacts with the substrate binding domain (SBD) through
salt bridges

2) The Heat Shock Protein 90:


a) Doesnt need ATP for its function
b) Has only 2 client proteins
c) Changes its conformation from open to
close
d) Uses Glutamate for the ATP binding
e) Has a Rossmann folding

5) The substrate binding domain (SBD) of Hsp70 is:


a) Responsible for the ATP hydrolisis
b) Is very conserved through its alpha region
c) Composed of a two-layered -sandwich
d) Has no regulatory motif
e) Usually interacts with the NBD through van der Waals interactions

3) The Heat Shock Protein 90:


a) Uses Aspartate for the ATP hydrolysis
b) Binds to many co-chaperones during
the protein folding
c) Is inhibited in most cancers
d) Forms a trimer in the cytoplasm
e) Is necessary for the eukaryotic cells
survival

6) Regarding Hsp70 co-chaperons:


a) DnaJ is a chaperon itself
b) GrpE is not a nucleotide exchange factor
c) DnaJ interacts with the NBD of the GrpE
d) GroEL is a co-chaperon that forms a ternary complex with Hsp70
and DnaK
e) All of them have a J domain

PEMS
7) In concern with heat shock proteins:
a) They function alone
b) Bacteria, eukaryotes, and yeast express chaperons
c) They can only be induced under stress conditions
d) Intracellular Hsp have immunological functions
e) Small Hsp's are ATP dependent
8) Heat Shock protein 60 (GroEL) structure is:
a) Bullet shaped
b) American football shaped
c) Cilinder shaped
d) Has no clear shape
e) Amorphous shaped
9) Regarding chaperones
a) They function alone
b) Bacterial chaperones are better characterized
c) They can only be induced under stress conditions
d) They are all ATP dependent
e) Any desease is realted with them

10) Chaperone GroEL:


a) It is a dimeric protein
b) Its monomers have 5 domains
c) Only unfolded proteins interact with the chaperone
d) Opposite rings work at the same time
e) Extension of its central channel allows protein encapsulation

THANK YOU FOR YOUR


ATTENTION

GroEL

STAMP MSA

GroEL

STAMP MSA

HSP 90

ATP CONFORMATION
N6
HSP 70: extended
1HJO
N6

HSP 90: bent

1AM1

HSP 90
PROTEIN FAMILY

CO-CHAPERONES
FUNCTION

HSP 70

Helps fold nascent polypeptide chains.

P23

Stabilazes HSP 90 association with clients.

AHA1

Stimulates HSP 90 activity.

HOP

Mediates interaction of HSP 70 and HSP 90.


Inhibits HSP 90 activity.

CDC37

Modulates interactions with kinases.


Inhibits HSP 90 activity.

IMMUNOPHILIN

Modulate interactions with hormone receptors.

HSP 90

MUTATIONS
2CG9

ASN-107

HB