BMB170ProteinsLecture5,Oct.10th

• ProteinFolding

• Chaperones

Protein Fates

Review: Hartl (2002) Science 295:1852 Kim..Hartl (2013) Ann Rev Biochem 82:323

Improving folding outcomes

Review: Hartl (2002) Science 295:1852

Molecularchaperones

• Agroupofunrelatedclassesofproteins

– Bindtoandstabilizeanunstableconformation

– Facilitatescorrectfateinvivo– Aren’tpartofthefinalstructure

• Possiblecorrectfates

– Folding,oligomericassembly,transporttoorganelle,disposalby

degradation

• Molecularchaperonesdon’tviolateAnfinsen’sselfassembly

principle

– Noinherentinformationinchaperoneaboutproteinsfinalfold

– Preventincorrectinteractionswithinandbetweennon-native

proteins

– Assistself-assembly

– Increasetheyieldbutnottherate(exceptforisomerases)

Chaperones

•Folding/stabilization-Hsp60/Chaperonin family‣GroEL/ES (Hsp60)‣TriC and thermosome

-Hsp70 family‣Activators

-Hsp90-sHsps-Hsp100-Trigger factor-Prefoldin-Calnexin/Calreticulin

•Folding catalyzers–Disulfide isomerases –Prolyl isomerases

•Targeting factors–ER Targeting

‣SRP/GET pathway–Nuclear targeting

‣Importin/Exportin•Assembly Factors

-Ribosome-3’ mRNA

3MainHSP

Families

• Conservedacross

kingdoms

• Discoveredbecauseof

heatshockinduction

– Alsoinducedfromother

stressesresultingin

accumulationof

unfoldedproteins

• Majorityexpressed

constitutively

– essentialforcellgrowth

Chaperone Summary

Review: Hartl (2002) Science 295:1852 Kim..Hartl (2013) Ann Rev Biochem 82:323

Chaperonin/Hsp60 family of proteins

Location Chaperone Roles

Prokaryotic cytosol GroEL/ GroES Protein folding, including elongation factor, RNA polymerase. Required for phage assembly

Mitochondria/ Chloroplasts

Hsp60/10 Cpn60/10

Folding and assembly of imported proteins

Archaebacterial cytosol

TF55 Thermosome

Binds heat-denatured proteins and prevents aggregation

Eukaryotic cytosol TCP-1, CCT, or TRiC

Folding of actin and tubulin; folds firefly luciferase in vitro

I. G

roEL

sub

fam

ilyII.

TCP

-1 s

ubfa

mily

• Foundinalldomains

• Structure

– Largecylindricaloligomers(tworings)

– Centralfoldingcavity

– Eachsubunithasthreedomains

GroupI:GroEL

• Firstidentified

– 1972Georgopoulos&KaiserandTanaka&Kakefuda

foundgenemutant“GroE”thatprotectedagainst

Lambdaphageinfection.

– GroEmutantshadaggregatedphageheads

– Rescuephagemutation

• λEmutantinphagemajorcapsidproteinwithlower

expressionlevels.

• λErescuedphageinfectivity.

• FirstPurified–HendrixandHohnetal• Activity–chaperoninassociatedwithRubisco(Barraclough

&Ellis1980)

– Ellis(Warwick,UK)firstused“molecularchaperone”

• Horwichhadtsmutantsinmitochondria

Mitochondrial Hsp60 identified from yeast mutant α143

Hsp60 mutations cause F1β to misassemble

In vitro translation with mitochondria

S35 labeled F1 β

(T)otal (A)queous (C)hloroform

Cheng..Hartl, Horwich (1989) Nature

F1β partitions to the chloroform phase with mutant Hsp60

Purification & EM of GroEL.

Hendrix 1979 JMB.

GroEL seen to have 7 fold symmetry.

Hendrix 1979 JMB.

Superpositions of an original image and the images rotated by 360/n degrees.

FirststructureofGroEL

• GroELisinbacterialcytoplasm

– Twostackedrings• Seven60kDsubunitseach(547aa)

• Arrangedwith7-foldsymmetry

– Cylinderwithcentralcavity

– Ringsarrangedback-to-backforming

interfaceacrossequatorialplane

• Invitrostudies– polypeptidesdilutedfromdenaturantshow

thatnon-nativeintermediatesbindtoGroEL

with1-2polypeptidesper14-mer

– Partofboundpolypeptideisincentralcavity

(EMstudies)

• GroES(10kD)bindsatoneendof

cylinder

Braig et al Nature (1994) 371: 578-86 (1grl)

GroELstructure

• Diameterofchannel:

– ~45Å

– Length:~146Å

– Totalvolume:~250,000Å3

• Spaceconsideration

– ~100kD(folded)or50-60kD(molten

globule)–F1βis60kD

– calculationassumesproteinspansthe

channelsofbothrings

– notbigenough(?)

• SuggestedfunctionofGroEL:

Anfinsencageforproteintobe

isolatedwhilefolding.Butresidues

affectingbindingarehydrophobic--

howdoesunfoldedproteinfalloff?

Braig et al Nature (1994) 371: 578-86 (1grl)

Saibil lab: Chen et al Nature Struct Biol (1994) 1: 838-42Review: Hartl Nature (1996) 381: 571-80

GroES binds to only one side of GroEL. Note changes in ring closest to GroES.

CryoEMreveals

asymmetricGroEL-

GroEScomplex

GroEL-GroES-(ADP)7 complex (1aon)

Sigler and Horwitch labs: Xu et al Nature (1997) 388: 741-60 (1aon)

Apical domains in GroEL ring nearest GroES move upwards to make a bigger cavity and that chamber becomes more hydrophilic. Trans ring doesn’t move compared with GroEL structure.

Cisring

80Å

Transring

71Å

Mobile loop was disordered in 6 of 7 subunits of GroES structure, but is ordered in all 7 subunits of GroES in GroEL-GroES complex.

Mobileloop

Sigler and Horwitch labs: Xu et al Nature (1997) 388: 741-60 (1aon)

GroES structure in GroEL-GroES complex

Accessible surface Hydrophobic

Hydrophilic

GroES

Cis

Trans

Internalcavityenlargesand

convertstohydrophilicupon

GroESbinding

Sigler and Horwitch labs: Xu et al Nature (1997) 388: 741-60 (1aon)

Group I Chaperonin

Mayer (2010) Mol Cell

Lin & Rye Crit Rev Biochem Mol Biol (2006) 41:211

• Unfolded protein binds in top ring - too big for channel

• ATP binding – allows GroES release from other side– it (or another GroES) binds to ring

with chain– cavity is bigger/hydrophilic - folding

starts• ATP hydrolysis

– weakens interaction between GroES and GroEL

• ATP binds to opposite ring – release of GroES– release of polypeptide from other ring

(~15s)

The GroEL-GroES reaction cycle

Thermosome structure• Discoveredin1991

• Archaeal group II chaperonin

• Structure from T. acidolphilum

• 2 subunits• Mg-ADP-AlF3 form

Ditzel..Steinbacher (1998) Cell 93:125 (1A6E)

Group II versus Group I

7 Subunits8 subunits

TRiC/CCT EM Structure

• Discovered in 1992 • 8 different subunits• Essential (10% of proteins) including actin• Conserved throughout eukaryotes• Organization, role of multiple subunits

Cong..Frydman, Chiu (2010) PNAS 107:4967

Group II Chaperonins

Mayer (2010) Mol Cell

• TRiC/CCT and thermosome

• 57-61kDa subunits

• Homo- or hetero-oligomers with 8 or 9 subunits

• 1:1 subunit interaction

OrganizationofTriC

Leitner..Chiu, Hartl, Aebersold, Frydman (2012) Structure 20:814

Yeast Bovine

Proteome-wide analysis of Chaperone-dependence

E. Coli

~2400 soluble proteins in proteome

~250 interact with GroEL (~400 in E. coli lacking TF, DnaJ and DnaK)

~85 proteins require GroEL (use ~75-80% GroEL)

Kerner..Hartl (2005) Cell 122:209

Can use

Need in vitro,not dependent

Require

Abundant soluble proteins are largely class I

Kerner..Hartl (2005) Cell 122:209

TIM-barrels predominate in class IIISCOP fold abbreviations: c.1, TIM β/α barrel;a.4, DNA/RNA binding 3-helical bundle; c.37, P loop containing nucleotide triphosphatehydrolases; c.67, PLP-dependent transferases; c.2, NAD(P) binding Rossmann fold domains; c.3, FAD/NAD(P) binding domain; c.23 flavodoxin-like; d.58, ferredoxin-like; c.47, thioredoxin fold; c.66, S-adenosyl-L-methionine-dependent methyltransferases.

TIM β/α barrel are 6.8% of lysate identified E.coli proteins.

Kerner..Hartl (2005) Cell 122:2098tim triosephosphate isomerase

GroEL associated proteins tend to be non-essential and low abundance…

…but the 13 essential proteins make GroEL essential.

Kerner..Hartl (2005) Cell 122:209

GroEL-deficient bacteria.

Mycoplasma and Ureaplasma genomes:

-Have orthologs of 25-40% of E.coli proteins generally.

-Have orthologs of 15-20% of Class III E.coli proteins.

Bacteria that lack GroEL have less proteins that require GroEL.

Kerner..Hartl (2005) Cell 122:209

BiP/Hsp70 class

• Foundinbacteria,mitochondria,cytoplasm,

andlumenofERineukaryoticcells

• Modulateconformationorassemblyofproteins

• InvolveATPbindingand/orhydrolysis

• Requireotherheatshockproteinsorother

cellularfactors

• Inducedbyaccumulationofunfoldedproteins

inappropriatecellularcompartment

Hsp70anddisease

• Drosophilamodelfor

neurodegeneralve

disease

• OverexpressionofHsp70

rescuesthephenotype

Bonini (2002) PNAS 99:16407-11

RoleofHsp70

• Duringstress–stabilizesproteinsagainstaggregation

• Normalgrowth

– Foldingofnewlysynthesizedproteins

– Subcellulartransportofproteinsandvesicles

– Formationanddissociationofcomplexes

– Degradationofunwantedproteins

• Shapesproteinhomeostasis

• Implicatedinanumberofdiseases

– Overexpressedinmanycancers

Hsp70 family of proteins

Location Chaperone Roles

Prokaryotic cytosol DnaK (50% identical to human) cofactors DnaJ, GrpE

Stabilizes newly synthesized polypeptides and preserves folding competence; reactivates heat-denatured proteins; controls heat-shock response

Eukaryotic cytosol SSA1, SSB1(yeast) Hsc/hsp70, hsp40 (mammalian)

Protein transport across organelle membranes; binds nascent polypeptides; dissociates clathrin from coated vesicles; promotes lysosomal degradation of cytosolic proteins

ER KAR2, BiP/Grp78 Protein translocation into ER

Mitochondria/ Chloroplasts

SSC1 ctHsp70

Protein translocation into mitochondria; Insertion of light-harvesting complex into thylakoid membrane

3copiesinE.coli,20copiesinyeast,

Gething & Sambrook Nature (1992) 355: 33-45

Roles of stress-70 proteins in eukaryotic cells

Cycle• Contain two domains

–NBD ~40kDa–SBD ~25kDa–Crosstalk occurs between domains

• Hsp70s are extremely slow ATPases (0.003 s-1)

–ATP Kd of 1nM • Co-chaperones regulate turnover

–J-proteins (Hsp40) stimulate hydrolysis

•~7-fold –NEFs – increase turnover

Structure has two parts: compact β-sandwich and extended structure of helices.

Zhu et al Science (1996) 272:1606-14 (1dkz)

Crystallized with substrate peptide (NRLLLTG) identified in phage display screen. (40-50% sequence identity with eukaryotic homologs)

Peptide binds in channel formed by loops from β-sandwich domain. Helical domain forms lid over peptide.

Highly conserved N-terminal domain with ATPase activity

Divergent C-terminal domain that binds unfolded proteins

Stress-70 proteins

Crystal structure of DnaK substrate-binding domain

Crystal structure of DnaK substrate-binding domain

Substrate peptide: NRLLLTG 7 H-bonds to peptide backbone

L4 of peptide buried in deep pocket.

Zhu et al Science (1996) 272:1606-14 (1dkz)

Hydrophobic Hydrophilic

Nucleotide binding domain

Hsc70 NBD (3hsc) DnaK NBD/GrpE (1dkg)

Harrison..Hartl, Kuriyan (1997) Science 276:431

Flaherty..McKay (1990) Nature 346:623

Full model

• ADP/apostructure

• NMRandspin-labelingtoconstrainmodel

• Reddomainlinkedtocontrolofpeptide

binding

Bertelsen..Gestwicki (2009) PNAS (2kho)

Allostericopening

Kityketal(2012)MolCell48:863-74:Qietal(2013)NSMB20:900-7

DnaK

(4B9Q)

Sse1

(4JNE)

Hsp70andoutcomes

Kampinga&Craig(2010)NatureRevMCB11:579

Hsp70linkedtodisease

• HighHsp70levelsassociatedwithbreast,

edometrial,oral,colorectal,prostatecancers

andcertainleukemias

• OverexperessioncaninduceTcelllymphoma

• Affectsapoptoticpathways

• Cancercellsbecome“addicted”toHsp70

• Assistsinresistancetochemotherapies

• Linkedtoneurodegenerativedisease

Evans..Gestwicki(2010)JMedChem

J-proteins

• DnaJ/Hsp40family

• Stabilizetheinteraction

withsubstrateproteins

• Manyhomologues

– 6inE.coli– 22inyeast

– >41inus

• Diversetissueand

organellelocalization

Qiu et al (2006) Cell Mol Life Sci 63:2560

J-proteindomain

architecture

Kampinga&Craig(2010)NatureRevMCB11:579

J domainGly–Phe-rich domainCTDI with ZFLRCTDIIDimerization domain

Putative CTDIICTDI lacking ZFLR

TransmembranedomainPutative CTDIUbiquitin-interactingmotifCoiled coilCTDI with HDAC-binding domainUnidentified motifZinc finger domain

Isu 1-binding domainSpliceosome-interaction domainThioredoxin boxExtracellular fragmentClathrin-bindingregion

Protein kinase domainGTP-binding site

Tetratricopeptiderepeat

HEPN domainSec63 domainSANT domain

ER signal peptide

Acetylatable Lys

Stretches not shown

ER retention peptideMitochondrial leader

Ribosome-binding region

0Yeast Human 100 200 300 400 500 600 Amino acids

Ydj1 DNAJA1

DNAJA3

Xdj1 DNAJA2, A4

DNAJB4, B5DNAJB11DNAJB9DNAJB2a, 2b

DNAJB6a, 6b

DNAJB12a, 12bDNAJB14a, 14bDNAJC18

DNAJC21Jjj1DNAJC24

DNAJC10DNAJC16

DNAJC26DNAJC27DNAJC3DNAJC7

DNAJC29DNAJC14DNAJC22

DNAJB13DNAJB3DNAJC13

DNAJC28DNAJC9DNAJC8DNAJC25

DNAJC11

DNAJC23Sec63

Zuo1

Mdj2

DNAJC1

DNAJC2DNAJC15DNAJC12DNAJC19DNAJC30

DNAJC4

Hlj1

Pam18

Jjj3DNAJC5, 5b, 5g

DNAJB8DNAJB7

Apj1Scj1Mdj1

DNAJB1Sis1

DNAJC20Jac1

Jem1

Jid1

Jjj2

DNAJC17Cwc23

Swa2

Djp1Caj1

Erj5

Promiscous client binding

Selective client binding

Client binding unclear

No client binding

Nature Reviews | Molecular Cell Biology

590/531

793

782

4,5794,306

702

2,2431,301

663/760

Cys-rich stretch

DNAJC6

Tensin-binding motif

668

913

1,311

UBA domain

REVIEWS

582 | AUGUST 2010 | VOLUME 11 www.nature.com/reviews/molcellbio

© 20 Macmillan Publishers Limited. All rights reserved10

J-domain proteins

Hdj1 J-domain Qian et al (1996) JMB 260:224(1hdj)

• J-domainisunifying

feature

• 70-aminoacids

• Threeclassesofproteins

– I&IIbindsubstrate

directly

• Nucleotidehydrolysis

andsubstratehandoff

HPDmotif

J-proteinfunclons

Kampinga&Craig(2010)NatureRevMCB11:579

Degradalon/assemblybyJ-protein

Kampinga&Craig(2010)NatureRevMCB11:579