BMB170 Proteins Lecture 5, Oct. 10Th

BMB170 Proteins Lecture 5, Oct. 10th

• Protein Folding • 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 Molecular chaperones • A group of unrelated classes of proteins – Bind to and stabilize an unstable conformation – Facilitates correct fate in vivo – Aren’t part of the final structure • Possible correct fates – Folding, oligomeric assembly, transport to organelle, disposal by degradation • Molecular chaperones don’t violate Anfinsen’s self assembly principle – No inherent information in chaperone about proteins final fold – Prevent incorrect interactions within and between non-native proteins – Assist self-assembly – Increase the yield but not the rate (except for isomerases) Chaperones

•Folding/stabilization •Folding catalyzers -Hsp60/Chaperonin –Disulﬁde isomerases family –Prolyl isomerases ‣GroEL/ES (Hsp60) •Targeting factors ‣TriC and –ER Targeting thermosome ‣SRP/GET pathway -Hsp70 family –Nuclear targeting ‣Activators ‣Importin/Exportin -Hsp90 •Assembly Factors -sHsps -Ribosome -Hsp100 -3’ mRNA -Trigger factor -Prefoldin -Calnexin/Calreticulin 3 Main HSP Families

• Conserved across kingdoms • Discovered because of heat shock induction – Also induced from other stresses resulting in accumulation of unfolded proteins • Majority expressed constitutively – essential for cell growth Chaperone Summary

Review: Hartl (2002) Science 295:1852 Kim..Hartl (2013) Ann Rev Biochem 82:323 Chaperonin/Hsp60 family of proteins

• Found in all domains • Structure – Large cylindrical oligomers (two rings) – Central folding cavity – Each subunit has three domains

Location Chaperone Roles

Prokaryotic cytosol GroEL/ GroES Protein folding, including elongation factor, RNA polymerase. Required for phage assembly Mitochondria/ Hsp60/10 Folding and assembly of imported

I. GroEL subfamily Chloroplasts Cpn60/10 proteins

Archaebacterial TF55 Binds heat-denatured proteins and cytosol Thermosome prevents aggregation Eukaryotic cytosol TCP-1, CCT, or Folding of actin and tubulin; folds firefly TRiC luciferase in vitro II. TCP-1 II. subfamily Group I: GroEL • First identified – 1972 Georgopoulos & Kaiser and Tanaka & Kakefuda found gene mutant “GroE” that protected against Lambda phage infection. – GroE mutants had aggregated phage heads – Rescue phage mutation • λE mutant in phage major capsid protein with lower expression levels. • λE rescued phage infectivity. • First Purified – Hendrix and Hohn et al • Activity – chaperonin associated with Rubisco (Barraclough & Ellis 1980) – Ellis (Warwick, UK) first used “molecular chaperone” • Horwich had ts mutants in mitochondria 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

F1β partitions to the chloroform phase with mutant Hsp60

Cheng..Hartl, Horwich (1989) Nature Purification & EM of GroEL.

Hendrix 1979 JMB. GroEL seen to have 7 fold symmetry.

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

Hendrix 1979 JMB. First structure of GroEL • GroEL is in bacterial cytoplasm – Two stacked rings • Seven 60 kD subunits each (547 aa) • Arranged with 7-fold symmetry – Cylinder with central cavity – Rings arranged back-to-back forming interface across equatorial plane • In vitro studies – polypeptides diluted from denaturant show that non-native intermediates bind to GroEL with 1-2 polypeptides per 14-mer – Part of bound polypeptide is in central cavity (EM studies) • GroES (10 kD) binds at one end of cylinder

Braig et al Nature (1994) 371: 578-86 (1grl) GroEL structure • Diameter of channel: – ~45 Å – Length: ~146 Å – Total volume: ~250,000 Å3 • Space consideration – ~100 kD (folded) or 50-60 kD (molten globule) – F1β is 60kD – calculation assumes protein spans the channels of both rings – not big enough (?) • Suggested function of GroEL: Anfinsen cage for protein to be isolated while folding. But residues affecting binding are hydrophobic -- how does unfolded protein fall off? Braig et al Nature (1994) 371: 578-86 (1grl) CryoEM reveals asymmetric GroEL- GroES complex

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

Saibil lab: Chen et al Nature Struct Biol (1994) 1: 838-42 Review: Hartl Nature (1996) 381: 571-80 GroEL-GroES-(ADP)7 complex (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.

Cis ring 80Å

Trans ring 71Å

Sigler and Horwitch labs: Xu et al Nature (1997) 388: 741-60 (1aon) GroES structure in GroEL-GroES complex

Mobile loop 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.

Sigler and Horwitch labs: Xu et al Nature (1997) 388: 741-60 (1aon) Accessible GroES surface Hydrophobic Hydrophilic Internal cavity enlarges and converts to hydrophilic upon GroES binding Cis

Trans

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

Mayer (2010) Mol Cell The GroEL-GroES reaction cycle

• 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)

Lin & Rye Crit Rev Biochem Mol Biol (2006) 41:211 Thermosome structure • Discovered in 1991 • 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

8 subunits 7 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

• TRiC/CCT and

thermosome

• 57-61kDa subunits • Homo- or hetero- oligomers with 8 or

9 subunits • 1:1 subunit

interaction

Mayer (2010) Mol Cell Organization of TriC

Yeast Bovine

Leitner..Chiu, Hartl, Aebersold, Frydman (2012) Structure 20:814 Proteome-wide analysis of Chaperone- dependence

E. Coli

~2400 soluble proteins in Can use proteome Need in vitro, ~250 interact with GroEL not dependent (~400 in E. coli lacking TF,

DnaJ and DnaK) Require

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

Kerner..Hartl (2005) Cell 122:209 Abundant soluble proteins are largely class I

Kerner..Hartl (2005) Cell 122:209 TIM-barrels predominate in class III

SCOP fold abbreviations: c.1, TIM β/α barrel; a.4, DNA/RNA binding 3-helical bundle; c.37, P loop containing nucleotide triphosphate hydrolases; 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.

8tim Kerner..Hartl (2005) Cell 122:209 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

• Found in bacteria, mitochondria, cytoplasm, and lumen of ER in eukaryotic cells • Modulate conformation or assembly of proteins • Involve ATP binding and/or hydrolysis • Require other heat shock proteins or other cellular factors • Induced by accumulation of unfolded proteins in appropriate cellular compartment Hsp70 and disease

• Drosophila model for neurodegeneralve disease • Overexpression of Hsp70 rescues the phenotype

Bonini (2002) PNAS 99:16407-11 Role of Hsp70

• During stress – stabilizes proteins against aggregation • Normal growth – Folding of newly synthesized proteins – Subcellular transport of proteins and vesicles – Formation and dissociation of complexes – Degradation of unwanted proteins

• Shapes protein homeostasis • Implicated in a number of diseases – Overexpressed in many cancers Hsp70 family of proteins

Location Chaperone Roles

Prokaryotic cytosol DnaK (50% identical Stabilizes newly synthesized polypeptides to human) and preserves folding competence; cofactors reactivates heat-denatured proteins; controls heat-shock response DnaJ, GrpE

Eukaryotic cytosol SSA1, SSB1(yeast) Protein transport across organelle Hsc/hsp70, hsp40 membranes; binds nascent polypeptides; (mammalian) dissociates clathrin from coated vesicles; promotes lysosomal degradation of cytosolic proteins

ER KAR2, BiP/Grp78 Protein translocation into ER

Mitochondria/ SSC1 Protein translocation into mitochondria; Chloroplasts ctHsp70 Insertion of light-harvesting complex into thylakoid membrane 3 copies in E. coli, 20 copies in yeast, Roles of stress-70 proteins in eukaryotic cells

Gething & Sambrook Nature (1992) 355: 33-45 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 Crystal structure of DnaK substrate-binding domain (40-50% sequence identity with eukaryotic homologs) Crystallized with substrate peptide (NRLLLTG) identified in phage display screen. Peptide binds in channel formed by loops from β- sandwich domain. Helical domain forms lid over peptide.

Stress-70 proteins

Highly conserved N- Divergent C- terminal domain with terminal ATPase activity domain that binds unfolded proteins

Structure has two parts: compact β-sandwich and extended structure of helices. Zhu et al Science (1996) 272:1606-14 (1dkz) Crystal structure of DnaK substrate-binding domain 7 H-bonds to peptide backbone Substrate peptide: NRLLLTG

L4 of peptide buried in deep pocket. Hydrophobic Hydrophilic

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

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

Flaherty..McKay (1990) Harrison..Hartl, Kuriyan (1997) Nature 346:623 Science 276:431 Full model

• ADP/apo structure • NMR and spin-labeling to constrain model • Red domain linked to control of peptide binding

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

Sse1 (4JNE)

DnaK (4B9Q)

Kityk et al (2012) Mol Cell 48:863-74: Qi et al (2013) NSMB 20:900-7 Hsp70 and outcomes

Kampinga & Craig (2010) Nature Rev MCB 11:579 Hsp70 linked to disease

• High Hsp70 levels associated with breast, edometrial, oral, colorectal, prostate cancers and certain leukemias • Overexperession can induce T cell lymphoma • Affects apoptotic pathways • Cancer cells become “addicted” to Hsp70 • Assists in resistance to chemotherapies • Linked to neurodegenerative disease

Evans..Gestwicki (2010) J Med Chem J-proteins

• DnaJ/Hsp40 family • Stabilize the interaction with substrate proteins • Many homologues – 6 in E. coli – 22 in yeast – >41 in us • Diverse tissue and organelle localization

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

Yeast Human 0 100 200300 400500 600 Amino acids

J domain Promiscous client binding Gly–Phe-rich domain Ydj1 DNAJA1 CTDI with ZFLR Xdj1 DNAJA2, A4 CTDII Apj1 Dimerization domain Scj1 CTDI lacking ZFLR Mdj1 DNAJA3 J-protein domain Putative CTDII Sis1 DNAJB1 Transmembrane DNAJB4, B5 domain DNAJB11 Putative CTDI Ubiquitin-interacting DNAJB9 motif DNAJB2a, 2b architecture Coiled coil Djp1 CTDI with HDAC- Caj1 binding domain DNAJB6a, 6b Unidentified motif DNAJB8 Zinc finger domain DNAJB7 Cys-rich stretch Isu 1-binding domain Erj5 Spliceosome- DNAJB12a, 12b interaction domain DNAJB14a, 14b Thioredoxin box DNAJC18 Extracellular fragment Selective client binding Clathrin-binding Jjj1 DNAJC21 590/531 region Jjj3 DNAJC24 UBA domain Tetratricopeptide DNAJC5, 5b, 5g repeat Jac1 DNAJC20 Tensin-binding motif Cwc23 DNAJC17 Protein kinase domain DNAJC10 793 GTP-binding site DNAJC16 782 HEPN domain Swa2 668 Sec63 domain SANT domain DNAJC6 913 Ribosome-binding DNAJC26 1,311 region DNAJC27 ER signal peptide DNAJC3 ER retention peptide DNAJC7 Mitochondrial leader Jem1 4,306 Acetylatable Lys DNAJC29 4,579 Stretches not shown DNAJC14 702 DNAJC22 Client binding unclear DNAJB13 DNAJB3 1,301 DNAJC13 2,243 Jid1 DNAJC28 DNAJC9 DNAJC8 DNAJC25 Jjj2 DNAJC11 No client binding Sec63 DNAJC23 663/760 DNAJC1 Zuo1 DNAJC2 Mdj2 DNAJC15 DNAJC12 Pam18 DNAJC19 DNAJC30 Hlj1 Kampinga & Craig (2010) Nature Rev MCB 11:579 DNAJC4

• J-domain is unifying feature • 70-amino acids • Three classes of proteins – I & II bind substrate directly • Nucleotide hydrolysis and substrate handoff

HPD motif

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

Kampinga & Craig (2010) Nature Rev MCB 11:579 Degradalon/assembly by J-protein

Kampinga & Craig (2010) Nature Rev MCB 11:579