Biología Estructural 4º Curso

CHAPERONES

MIREN AIZPIRI

KANISHKA BHAMBI

JAIME CANO

INDEX

INTRODUCTION

HSP 60 (GroEL)

HSP 70

HSP 90

CONCLUSIONS

CHAPERONES

Definition: An abundant class of proteins thatassist in the correct folding and maturation of other cellular proteins.

Dont participate in the final mature structure.

Have little specificity for theirsubstrates.

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

HSP60

GroEL

HSP 60 INTRODUCTION

First large oligomeric chaperone (1970s)

Essential for cell survival

50% of proteins interact with it 30% are unable to fold whithout it

GroES

Highly conserved Present in mythocondria and

citoplasm Protein folding and transport DNA metabolism Apoptosis

PDB: 1AON

GroEL STRUCTURE

Each subunit

Apical domain

Intermediate domain

Ecuatorial domain

GroES

GroEL

14 identical subunits arranged in two heptamericrings stacked back-to-back

PDB: 1AON

GroEL APICAL DOMAIN

Polypeptide binding site GroES binding site

Root: ScopFold: 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 betweenApical and Equatorialdomains

Root: ScopFold: Alpha and beta proteins (a+b)Class: GroEL-intermediate domain like Superfamily: GroEL-intermediate domain like

PDB: 1AON

GroEL ECUATORIAL DOMAIN

Root: ScopFold: GroEL equatorial domain-likeClass: All alpha proteinsSuperfamily: Equatorial domain-like

ATP binding site Inter and intra ring contacts

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

PDB: 1AONBuckle AM et al. A structural model for GroEL-polypeptiderecognition. Proc Natl Acad Sci U S A . 1997 Apr;94(8):35715.


Groel T state side view PDB: 1OEL


Y199S201Y203F204


L234L237L259V263V264

GroEL PROTEIN ENCAPSULATION

T State RS2 State RS-open State

ATP

RS1 State R-ES State

PDB: 1AONPDB: 1CXK PDB: 4AAQ PDB: 4AAS PDB: 2C7D


Left side view Right side viewFrontal view

PDB white: 1CXK / PDB yellow: 4AAS

E255-K207

R197-E386

PDB: 1CXK

E255-K452

K80 -E386

PDB: 4AAQ

PDB: 4AAS

K80 -E386

PDB: C2D7


K80

R197K207


E386

K245


R452

GroEL ATP BINDING POCKET

D398

PDB: 1AON D398A


I150S151

T30L31G32P33

K51D52G53

D87G88T89T90T91


I454I493D495

D398G414G415G416

GroEL HYDROPHYLIC SURFACE

Hydrophylic inner ring surface PDB: 1AON

K4

K42E61K75D85

D196R197


K266E252D253E255E257

K277D283R285K286K327D328

D359E363K364E367R368K371

K380E386K393


R404E408

D523


Cluster: 1 ( 1AONA & 4V40a ) Sc 8.43 RMS 1.41Cluster: 2 ( 1IOKA & 1SJPA ) Sc 5.71 RMS 1.88Cluster: 3 ( 1AONA 4V40a & 1IOKA 1SJPA ) Sc 5.92 RMS 2.68

GroEL STAMP

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

Cluster: 1 ( 1oelA & 1IOKA ) Sc 8.20 RMS 1.16Cluster: 2 ( 1SJPA & 1oelA 1IOKA ) Sc 7.86 RMS 2.11Cluster: 3 ( 4V40A & 1SJPA 1oelA 1IOKA ) Sc 5.80 RMS 2.53

GroEL STAMP

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

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 gram-negative 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.

Proof for the endosymbiont

theory

HSP 70 EVOLUTION

Two large divisions:

o prokaryotic DnaK proteins together with the plastidal and mitochondrial sequences

o eukaryotic nucleo/cytoplasmaticand 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

N-terminal nucleotide binding domain (NDB) ATPase activity

C-terminal substrate binding domain (SBD)

If the NBD is bound to ADP

However, when ATP is bound to the NBD

NRLLLTG interacts with high affinity

Substrate binds significantly more weakly

STRUCTURE

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

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.

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.

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

IA

IIA

IIB

PDB:1S3X

HSP 70 Human Hsp70 - NBD

PDB:1SX3

PDB:1S3X

HSP 70 SEQUENCE ALIGNMENT

PDB:1SX3

PDB:1S3X

Highly conserved amino acids: T13, K71, and T204

HSP 70 Human Hsp70 - NBD

PDB:1SX3

New metal binding motif at the junction between: - a beta sheet (190-

225) - and alpha helix (230-250)- Close to the catalytic site

PDB:1S3X

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

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 DnaK - SBD

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

Existence of considerable structural variability within the SBD region

Sc = 3.75RMSD = 1.61

PDB:4PO2,magenta

PDB:1DKZ,blue

HSP 70 SBD REGION

Structural alignment Alpha domains of the SBD of Rat Hsc70 and our Hsp70

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

Sc = 0.51RMSD = 2.23

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 = 5.80RMSD = 0.48

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

HSP 90 INTRODUCTION

Comprises 1-2% of total cellular protein content.

In vertebrates: HSP 90 : inducible HSP 90 : constitutive Grp94: in the endoplasmic reticulum TRAP1: in the mitochondria

Principal clients: Nuclear hormone receptors Protein kinases

ESSENTIAL for the viability of the cell

HSP 90 EVOLUTION

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

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.

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: 1 210 272 629 732

Domains: N-terminal CR Middle

N-terminal CR Middle

Function: ATP binding Client binding DimerizationATP binding

HSP 90 MECHANISM OF ACTION

2O1V

CLOSE

ATP binding

ATP hydrolysis = ADP + Pi

2CG9

OPEN

2IOQ


Root: Scop

Class: Alpha + Beta

Fold: ATPase domain of HSP90 chaperone/DNA topoisomerase II/histidine kinase

Eigh-strandedantiparallel beta sheetcovered on one face bynine alpha helices, four

of them are 310 type.

PDB: 1AM1

E. Coli DNA Gyrase B and yeast HSP90 superimposition.


Score: 3.85RMS: 2.13

1AJ6 1AM1

They have a commonancestor, but divergedearly in evolution.

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


1YET 1A4H

Human and yeast superimposition



H9

H2H4 L1

H5

L2

H2

L2

H5

H4

N-terminal domain

L2 and H5 formthe lid thatconstricts thepocket entrance.


1AM1

H9

H2H4 L1

H5

L2

Superimpositionof the N-terminaldomain in openand closeconformation


1YES 1YER

L2

H5

HSP 90 N-TERMINAL ATP POCKET

15 A deep.

A half of the 17 aa lining itsinterior are hydrophobic, a quarter polar, and a quarter charged.

PDB: 1AM1

HSP 90 N-TERMINAL ATP POCKET

2CG9

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 higherthan 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 additionalHBs and increases ATPase activity.

HSP 90 MUTATIONS

HSP 90 CLUSTAL N-TERMINAL

Glu: ATP hydrolysis

Asp, Asn: ATP binding

GxxGxG motif of the lid.

Different HSP90 isoforms sequence alignment

HSP 90 CLUSTAL N-TERMINAL

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

3 short helices arrangedin a right-handed coil

PDB: 3PRY

sandwich

sandwich

Human and yeast superimposition

HSP 90 MIDDLE DOMAIN


1HK7

3PRY

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 steroidhormone 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.

The loop between strandsb and c contains a secondshort helix (H2).

H3, H4 and H5.

H1

H2

H5H4

H3

HSP 90 CLUSTAL C-TERMINAL

ANMERIMKA: binding of glucocorticoid receptor.

(M)EEVD motif: binding of TPR-domaincontaining 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 groovethat interacts with the (M)EEVD motif.

1ELR

HSP 90 (M)EEVD MOTIF

1ELR

(M)EEVD MOTIF

This motif is also conserved in the HSP 70 C-terminal domain.

HSP 90 CANCER

Ansa ring

Benzoquinone

Carbamat group

Geldanamycin

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.

Inhibition of ATP binding and hydrolysis

Degradation of oncogenicHSP 90 clients by proteasome

Superimposition of Geldanamycin-bound N-terminaldomain with ATP-bound N-terminal domain.

HSP 90 GELDANAMYCIN

1YET 1AM1

ATP in redGeldanamycin in blue

Score: 9.06RMSD: 0.71

HSP 90 GELDANAMYCIN

5 HB:

- with Lys-112

- with Lys -58

- with Asp-93

- with Thr-184

- with Phe-138

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

4PO2Crystal 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 The substrate binding domain of dnak in complex with a substrate peptide, determined from type 1 native crystals

1UD0 CRYSTAL STRUCTURE OF THE C-TERMINAL 10-kDA SUBDOMAIN OF HSC70

3FE1Crystal structure of the human 70kDa heat shock protein 6 (Hsp70B') ATPase domain in complex with ADP and inorganic phosphate

3GDQCrystal structure of the human 70kDa heat shock protein 1-like ATPase domain in complex with ADP and inorganic phosphate

3I33Crystal structure of the human 70kDa heat shock protein 2 (Hsp70-2) ATPase domain in complex with ADP and inorganic phosphate

3JXU Crystal structure of the human 70kDa heat shock protein 1A (Hsp70-1) ATPase domain in complex with ADP and inorganic phosphate

PDB Ids

PDB ID DESCRIPTION

1AM1 ATP BINDING SITE IN THE HSP90 MOLECULAR CHAPERONE

1AJ6NOVOBIOCIN-RESISTANT MUTANT (R136H) OF THE N-TERMINAL 24 KDA FRAGMENT OF DNA GYRASE B COMPLEXED WITH NOVOBIOCIN AT 2.3 ANGSTROMS RESOLUTION

1YETGELDANAMYCIN BOUND TO THE HSP90 GELDANAMYCIN-BINDING DOMAIN

1A4HSTRUCTURE OF THE N-TERMINAL DOMAIN OF THE YEAST HSP90 CHAPERONE IN COMPLEX WITH GELDANAMYCIN

3PRYCrystal structure of the middle domain of human HSP90-beta refined at 2.3 A resolution

1HK7 MIDDLE DOMAIN OF HSP90

2CG9CRYSTAL STRUCTURE OF AN HSP90-SBA1 CLOSED CHAPERONE COMPLEX

2O1V Structure of full length GRP94 with ADP bound

1YERHUMAN HSP90 GELDANAMYCIN-BINDING DOMAIN, "CLOSED" CONFORMATION

PDB Ids

PDB ID DESCRIPTION

1YESHUMAN HSP90 GELDANAMYCIN-BINDING DOMAIN,

"OPEN" CONFORMATION

1HJO ATPase domain of human heat shock 70kDa protein 1

1AONCRYSTAL STRUCTURE OF THE ASYMMETRIC

CHAPERONIN COMPLEX GROEL/GROES/(ADP)7

1XCKCrystal structure of apo GroE

4AAS ATP-triggered molecular mechanics of the chaperonin GroEL

4AAQ ATP-triggered molecular mechanics of the chaperonin GroEL

2C7DFitted coordinates for GroEL-ADP7-GroES Cryo-EM complex

(EMD-1181

1IOKCRYSTAL STRUCTURE OF CHAPERONIN-60 FROM

PARACOCCUS DENITRIFICANS

4V40Crystal Structure of the Chaperonin Complex

Cpn60/Cpn10/(ADP)7 from Thermus Thermophilus

REFERENCES

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

Zhang P, Leu JI-J, Murphy ME, George DL, Marmorstein R (2014) Crystal Structure of the Stress-Inducible Human Heat Shock Protein 70 Substrate-Binding Domain in Complex with Peptide Substrate. PLoS ONE 9(7): e103518. doi:10.1371/journal.pone.0103518

Sriram, M., Osipiuk, J., Freeman, B. C., Morimoto, R. I., & Joachimiak, A. (n.d.). Human Hsp70 molecular chaperone binds two calcium ions within the ATPase domain, 2325.

Mayer, M. P., & Bukau, B. (2005). Hsp70 chaperones: Cellular functions and molecular mechanism. Cellular and Molecular Life Sciences, 62, 670684.

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.

Rensing, S. a. (1994). Journal of Molecular Evolution, 8086.

Saibil H. Chaperone machines for protein folding, unfolding and disaggregation. Nat Rev Mol Cell Biol [Internet]. Nature Publishing Group; 2013;14(10):63042. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24026055

Doyle SM, Genest O, Wickner S. Protein rescue from aggregates by powerful molecular chaperone machines. Nat Rev Mol Cell Biol [Internet]. NaturePublishing Group; 2013 Oct [cited 2015 Mar 5];14(10):61729. Available from: http://www.ncbi.nlm.nih.gov/pubmed/24061228

Schmitt E, Gehrmann M, Brunet M, Multhoff G, Garrido C. Intracellular and extracellular functions of heat shock proteins: repercussions in cancertherapy. J Leukoc Biol [Internet]. 2007 Jan [cited 2015 Mar 5];81(1):1527. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16931602

Prodromou C, Roe SM, OBrien R, Ladbury JE, Piper PW, Pearl LH. Identification and Structural Characterization of the ATP/ADP-Binding Site in theHsp90 Molecular Chaperone. Cell [Internet]. 1997 Jul;90(1):6575. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0092867400803141

Ali MMU, Roe SM, Vaughan CK, Meyer P, Panaretou B, Piper PW, et al. Crystal structure of an Hsp90nucleotidep23/Sba1 closed chaperonecomplex. Nature [Internet]. 2006 Apr 20 [cited 2015 Mar 5];440(7087):10137. Available from: http://www.nature.com/doifinder/10.1038/nature04716

Li J, Soroka J, Buchner J. The Hsp90 chaperone machinery: conformational dynamics and regulation by co-chaperones. Biochim Biophys Acta [Internet]. Elsevier B.V.; 2012 Mar [cited 2015 Feb 11];1823(3):62435. Available from: http://www.ncbi.nlm.nih.gov/pubmed/21951723

Pearl LH, Prodromou C. Structure and mechanism of the Hsp90 molecular chaperone machinery. Annu Rev Biochem [Internet]. 2006 Jan [cited 2015 Mar 5];75:27194. Available from: http://www.ncbi.nlm.nih.gov/pubmed/16756493

Obermann WMJ, Sondermann H, Russo AA, Pavletich NP, Hartl FU. In Vivo Function of Hsp90 Is Dependent on ATP Binding and ATP Hydrolysis. 1998;143(4):90110.

Chiosis G, Vilenchik M, Kim J, Solit D. Hsp90: the vulnerable chaperone. Drug Discov Today [Internet]. 2004 Oct 15;9(20):8818. Available from: http://www.ncbi.nlm.nih.gov/pubmed/15475321

REFERENCES

Tatokoro M, Koga F, Yoshida S, Kihara K. Review article : HEAT SHOCK PROTEIN 90 TARGETING THERAPY : STATE OF THE ART AND FUTURE PERSPECTIVE. 2015;4858. Stebbins CE, Russo A a, Schneider C, Rosen N, Hartl FU, Pavletich NP. Crystal Structure of an Hsp90Geldanamycin Complex: Targeting of a Protein Chaperone by an

Antitumor Agent. Cell [Internet]. 1997 Apr 18;89(2):23950. Available from: http://linkinghub.elsevier.com/retrieve/pii/S0092867400802032 Whitesell L, Lindquist SL. HSP90 and the chaperoning of cancer. Nat Rev Cancer [Internet]. 2005 Oct [cited 2014 Nov 4];5(10):76172. Available from:

http://www.ncbi.nlm.nih.gov/pubmed/16175177 Huang L-H, Wang H-S, Kang L. Different evolutionary lineages of large and small heat shock proteins in eukaryotes. Cell Res [Internet]. 2008 Oct [cited 2015 Mar

5];18(10):10746. Available from: http://www.ncbi.nlm.nih.gov/pubmed/18766171 Ns D, Aa A, Ss U. Hsp: Evolved and Conserved Proteins, Structure and Sequence Studies. Int J Bioinforma Res [Internet]. 2010 Dec 30;2(2):6787. Available from:

http://bioinfopublication.org/viewhtml.php?artid=BIA0001366 Zhang X, Kelly JW. Chaperonins resculpt folding free energy landscapes to avoid kinetic traps and accelerate protein folding. J Mol Biol [Internet]. Elsevier Ltd;

2014;426(15):27368. Available from: http://dx.doi.org/10.1016/j.jmb.2014.06.001 Brocchieri L, Karlin S. Conservation among HSP60 sequences in relation to structure, function, and evolution. Protein Sci. 2000;9:47686. Buckle AM, Zahn R, Fersht AR. A structural model for GroEL-polypeptide recognition. Proc Natl Acad Sci U S A [Internet]. 1997 Apr 15 [cited 2015 Mar 7];94(8):35715.

Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=20480&tool=pmcentrez&rendertype=abstract Chen DH, Madan D, Weaver J, Lin Z, Schrder GF, Chiu W, et al. XVisualizing GroEL/ES in the act of encapsulating a folding protein. Cell. 2013;153. Clare DK, Vasishtan D, Stagg S, Quispe J, Farr GW, Topf M, et al. ATP-triggered conformational changes delineate substrate-binding and -folding mechanics of the

GroEL chaperonin. Cell [Internet]. 2012 Mar 30 [cited 2014 Dec 20];149(1):11323. Available from: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3326522&tool=pmcentrez&rendertype=abstract

Coyle JE, Jaeger J, Gross M, Robinson C V, Radford SE. Structural and mechanistic consequences of polypeptide binding by GroEL. Fold Des. 1997;2:R93104. Georgescauld F, Popova K, Gupta AJ, Bracher A, Engen JR, Hayer-Hartl M, et al. GroEL/ES chaperonin modulates the mechanism and accelerates the rate of TIM-barrel

domain folding. Cell [Internet]. Elsevier Inc.; 2014;157(4):92234. Available from: http://dx.doi.org/10.1016/j.cell.2014.03.038 Henderson B, Fares M a., Lund P a. Chaperonin 60: A paradoxical, evolutionarily conserved protein family with multiple moonlighting functions. Biol Rev.

2013;88:95587. Horwich AL, Fenton W a, Chapman E, Farr GW. Two families of chaperonin: physiology and mechanism. Annu Rev Cell Dev Biol. 2007;23:11545. Ranson N a, Clare DK, Farr GW, Houldershaw D, Horwich AL, Saibil HR. Allosteric signaling of ATP hydrolysis in GroEL-GroES complexes. Nat Struct Mol Biol.

2006;13(2):14752. Ryabova N a, Marchenkov V V, Marchenkova SY, Kotova N V, Semisotnov G V. Molecular Chaperone GroEL / ES : Unfolding and Refolding Processes. 2013;78(13). 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. Hartl FU, Bracher A, Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis. Nature [Internet]. Nature Publishing Group; 2011 Jul 21 [cited 2014 Jul

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-terminalb) Requires Calcium for its functionc) Is absent in non-stress conditionsd) Is not conserved among different speciese) Is an essential protein in prokariotes

2) The Heat Shock Protein 90:a) Doesnt need ATP for its functionb) Has only 2 client proteinsc) Changes its conformation from open to close d) Uses Glutamate for the ATP bindinge) Has a Rossmann folding

3) The Heat Shock Protein 90:a) Uses Aspartate for the ATP hydrolysisb) Binds to many co-chaperones during the protein foldingc) Is inhibited in most cancersd) Forms a trimer in the cytoplasme) Is necessary for the eukaryotic cells survival

4) The nucleotide binding domain (NBD) of the Hsp70 chaperon family:a) Has ATPase activity b) Consists of three globular domainsc) When is bound to ADP interacts more weakly with the substrated) When is bound to ATP interacts with high affinity with the substratee) Always interacts with the substrate binding domain (SBD) through salt bridges

5) The substrate binding domain (SBD) of Hsp70 is: a) Responsible for the ATP hydrolisisb) Is very conserved through its alpha region c) Composed of a two-layered -sandwichd) Has no regulatory motif e) Usually interacts with the NBD through van der Waals interactions

6) Regarding Hsp70 co-chaperons: a) DnaJ is a chaperon itself b) GrpE is not a nucleotide exchange factorc) DnaJ interacts with the NBD of the GrpEd) GroEL is a co-chaperon that forms a ternary complex with Hsp70 and DnaKe) All of them have a J domain

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

PEMS

10) Chaperone GroEL:a) It is a dimeric proteinb) Its monomers have 5 domainsc) Only unfolded proteins interact with the chaperoned) Opposite rings work at the same timee) Extension of its central channel allows protein encapsulation

THANK YOU FOR YOUR

ATTENTION

GroEL STAMP MSA

HSP 90 ATP CONFORMATION

HSP 70: extended

HSP 90: bent

1HJO

1AM1

N6

N6

HSP 90 CO-CHAPERONES

PROTEIN FAMILY FUNCTION

HSP 70 Helps fold nascent polypeptide chains.

P23 Stabilazes HSP 90 association with clients.

AHA1 Stimulates HSP 90 activity.

HOPMediates interaction of HSP 70 and HSP 90.Inhibits HSP 90 activity.

CDC37Modulates interactions with kinases.Inhibits HSP 90 activity.

IMMUNOPHILIN Modulate interactions with hormone receptors.

HSP 90 MUTATIONS

2CG9

ASN-107

HB

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Biología Estructural 4º Curso