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