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