BMB170ProteinsLecture6,Oct.12th
• Chaperones(cont)• Mo=fs/Domains
Chaperone Summary
Review: Hartl (2002) Science 295:1852 Kim..Hartl (2013) Ann Rev Biochem 82:323
Kampinga&Craig(2010)NatureRevMCB11:579Kityk..Sinning&Mayer(2012)MolCell48:863Kim..Hartl(2013)AnnRevBiochem82:323
StructuralCycle
Co-chaperones
TAtarge=ngispartofasurveillancesysteminthecytosolforhydrophobicdomains
Fungal Metazoan(Apesincluded!)
TA Protein
Chaperones
Sgt2
Get5
Get4
Get3
ER
Fast
Fast
TA Protein
Chaperones
SGTA
Ubl4A
TRC35
TRC40ER
Fast
Fast
Bag6
Trash
Mislocalized MPs
Bag6/Bat3/Scythe- Bag6 arrives with metazoans
(tissues in germ layers, neurons, gastrulation)
- Implicated in apoptosis, DNA damage response, cell cycle progression and immunoregulation
- TRC35 & TRC40 are the most conserved
Human
Sea Urchin
Nematodes
Fruit Fly
Sea Snails
Earthworms
Sea Anemone
Fresh Water Polyp
Trichoplax
Sponge
Capsaspora
Brewer’s yeast
Trichonomas
Plasmodiophora
Arabidopsis
Dictyostelium
C
Fungi
Amoebazoa
Excavata
Rhizaria
Plantae
Metazoa Bag6SGTA
Sgt2
TRC40
Get3
TRC35
Get4
Ubl4AUbl4A
Get5Get5
Bag6iscomplexMEPNDSTSTAVEEPDSLEVLVKTLDSQTRTFIVGAQMNVKEFKEHIAASVSIPSEKQRLIYQGRVLQDDKKLQEYNVGGKVIHLVERAPPQTHLPSGASSGTGSASATHGGGSPPGTRGPGASVHDRNANSYVMVGTFNLPSDGSAVDVHINMEQAPIQSEPRVRLVMAQHMIRDIQTLLSRMECRGGPQPQHSQPPPQPPAVTPEPVALSSQTSEPVESEAPPREPMEAEEVEERAPAQNPELTPGPAPAGPTPAPETNAPNHPSPAEYVEVLQELQRLESRLQPFLQRYYEVLGAAATTDYNNNHEGREEDQRLINLVGESLRLLGNTFVALSDLRCNLACTPPRHLHVVRPMSHYTTPMVLQQAAIPIQINVGTTVTMTGNGTRPPPTPNAEAPPPGPGQASSVAPSSTNVESSAEGAPPPGPAPPPATSHPRVIRISHQSVEPVVMMHMNIQDSGTQPGGVPSAPTGPLGPPGHGQTLGQQVPGFPTAPTRVVIARPTPPQARPSHPGGPPVSGTLQGAGLGTNASLAQMVSGLVGQLLMQPVLVAQGTPGMAPPPAPATASASAGTTNTATTAGPAPGGPAQPPPTPQPSMADLQFSQLLGNLLGPAGPGAGGSGVASPTITVAMPGVPAFLQGMTDFLQATQTAPPPPPPPPPPPPAPEQQTMPPPGSPSGGAGSPGGLGLESLSPEFFTSVVQGVLSSLLGSLGARAGSSESIAAFIQRLSGSSNIFEPGADGALGFFGALLSLLCQNFSMVDVVMLLHGHFQPLQRLQPQLRSFFHQHYLGGQEPTPSNIRMATHTLITGLEEYVRESFSLVQVQPGVDIIRTNLEFLQEQFNSIAAHVLHCTDSGFGARLLELCNQGLFECLALNLHCLGGQQMELAAVINGRIRRMSRGVNPSLVSWLTTMMGLRLQVVLEHMPVGPDAILRYVRRVGDPPQPLPEEPMEVQGAERASPEPQRENASPAPGTTAEEAMSRGPPPAPEGGSRDEQDGASAETEPWAAAVPPEWVPIIQQDIQSQRKVKPQPPLSDAYLSGMPAKRRKTMQGEGPQLLLSEAVSRAAKAAGARPLTSPESLSRDLEAPEVQESYRQQLRSDIQKRLQEDPNYSPQRFPNAQRAFADDP
UBLDomain–Caspase-3site–Nuclearlocaliza=onsignal–BAGdomain–Phosphorylated
AKABat3(HLA-B-associatedtranscript3)orScytheTRC35
Ubl4A
BAG(Bcl2-associatedathanogene)FamilyProteins
• Firstidentifiedin1995asanti-celldeath(athanos-greekforimmortal)protein(Takayamaetal.Cell80:279-284)
• BAGdomain– C-terminus– Hsp/Hsc70bindingattheATPase
domainandregulation
• DiversityofadditionaldomainsattheN-terminus– Bag1andBag6:ubiquitin-like(Ubl)
domain– Bag1L:Nuclearlocalizationsequence– Bag3:WWdomainand–PXXP–
repeat
Rosatietal.CellDeathandDisease(2011)2,e141.
BagDomainsBag1 Bag2 Bag3 Bag5Bag4
Arakawaetal.Structure(2010)18:309-319
Briknarovaetal.JBC(2002)277:31172-31178
PDBID:1UK5
Xuetal.Nat.Struc.Mol.Biol.(2008)15:1309-1317
Sondermannetal.Science(2001)291:1553-1557
Get5-Dimer
Bag6-BAG
Ubl4A-dimerizationBag6/Ubl4Aheterodimerdomains
Mock, Chartron, Zaslaver, Xu, Ye & Clemons (2015) PNAS
A ‘mock’ BAG domain• Contains some of the residues
involved in Hsc70 binding
• Does not form a complex in solution with Hsc70-NBD
• Cannot inhibit Hsc70 protein folding
• Does not pulldown Hsc70 from lysate
Bag1 (1HX1)
E219
E212
R237
α1
α2
α3
K238
D222
Q245
Bag6-BAG
α1
α2
α3E1065
R1100Q1101
Q1109
Bag1 PQEEVELKKLKHLEKSVEKIADQLEELNKELTGIQQGFLPKDLQAEALCKLDRRVKA
TIEQFMKILEEIDTLIL PENFK D
Bag5 SHKAVWNVLGNLSEIQGE VLSFGNRTDK NYIR
LEELLTKQLLALDA VD PQGEE K
Bag6
LSEAVSRAAKAAGARPLTSPESLSRDLEAPEVQESBag1 SR L KRKGLVKKVQAFLAECDTVEQNBag5 CK A ARKQAVRLAQNILSYLDLKS--Bag6 YRQQLRSDIQK
-----
--
----- -- -
----
α1
α2
Bag1-BAG
α1
α3
α2 α3
Bag1-BAG
Bag6-BAG
1109257442
209397--------------------
0
5
10
15
20
25
30
35
40
45
50
0 30 60 90 120 150
180
210 240
ß-ga
lact
osid
ase
activ
ity (%
)
Time (min)
Negative Control Hsc70+Hdj1 Hsc70+Hdj+Bag1 Hsc70+Hdj1+Bag6-BAGHsc70+Hdj1+Bag6EBAG-Ubl4A Hsc70+Hdj1+SGTA+Bag6-BAG Hsc70+Hdj1+SGTA+Bag6EBAG-Ubl4A
Hop – Hsp Organizing ProteinHSP70peptide:DDVD-COOH• Contains three TPR
domains (34 aa repeat) • Carboxylate clamp • TPR1 binds C-terminus of
Hsp70 • TPR2A binds Hsp90 • Hands off from Hsp70 to
Hsp90 • Other example is CHIP Other examples include protein
phosphatase 5 (Ppp5) which binds and phosphorylates Hsp90 or CHIP which targets Hsp70/90 for degradation. Scheufler et al (2000) Cell (1elw)
Cell200
Figure 1. Interaction of the TPR1 Domain of Hop with Hsc70/Hsp70
(A) Organization of the Hop coding region. The boundaries of the TPR1, TPR2A, and TPR2B domains are indicated.(B) Calorimetric isothermal titration measurement of the interaction of the Hop TPR1 domain with the C-terminal 25 kDa domain of Hsp70(C70). Top part: incremental heat effect upon titration of C70 (180 ⇥M) with TPR1 (3 mM) at 25�C. Lower part: integrated heat effects normalizedto the amount of injected TPR1 and fitted curve based on a 1:1 binding model. Constants determined were KD ⇤ 15 ⇥M, stoichiometry N ⇤ 0.87.(C) Thermodynamic binding constants (KD) for the interactions of TPR1 with C70 and peptides comprising the last 12 residues of Hsp70 andHsc70 (upper part), and for the binding of TPR2A to C90 and a Hsp90 pentapeptide (lower part). Titrations were performed at 25�C by injectingproteins or peptides into a solution containing TPR domains. Stoichiometry factors (N) typically ranged between 0.9 and 1.1. The ITCmeasurements were highly reproducible, with standard deviations for triplicate measurements ranging between 5% and 10%.
Hsp70 and Hsp90 family members known to interact (amino acids 629–732) bound to TPR2A with an affinityof 6 ⇥M and a stoichiometry factor close to one (Figurewith TPR proteins.1C). A peptide comprising only the last five residues ofHsp90 still bound to TPR2A with an affinity of 11 ⇥M
Results and Discussion (Figure 1C).Thus, the binding of TPR1 to C70 can be fully de-
Peptide Binding by Hop TPR Domains scribed by the interaction of TPR1 with a 12-mer C-ter-Hop has previously been shown by sequence analysis minal peptide of Hsp70, while the interaction of TPR2Ato contain two clusters of TPR elements (Chen et al., with C90 is essentially mediated by the five C-terminal1996; Lassle et al., 1997). Utilizing limited proteolysis to residues ofHsp90. Theaffinitiesmeasuredare compara-define the domains of human Hop, three stable frag- ble to those determined for the interaction of SH3 do-ments could be identified: TPR1, TPR2A, and TPR2B mains with their peptide ligands (Kuriyan and Cowburn,(Figure 1A; C. S. and I. M., unpublished data). We ex- 1997).pressed and purified fragments corresponding to theN-terminal TPR1 domain (amino acids 1–118) and the Structures of the TPR Domain–Peptide Complexesmiddle domain TPR2A (amino acids 223–352). Isother- We crystallized the TPR1 and TPR2A domains of Hopmal calorimetric (ITC) measurements (Wiseman et al., in complex with their respective peptide partners and1989) demonstrated that a 25 kDa fragment of human solved the high resolution crystal structures of the twoHsp70 (C70), retaining the substrate binding domain and complexes (see Experimental Procedures). Both TPRthe authentic C terminus of Hsp70, bound to TPR1 with domains form a meander of seven ⌅ helices (Figures 2Aan affinity of 15 ⇥M and a stoichiometry factor (N) of and 2E) which are arranged in a head to tail mannerclose to 1 (Figures 1B and 1C). The same affinities were similar to that of the peptide-free TPR domain of PP5obtained with 12-mer peptides of the nearly identical C (Das et al., 1998). Compared to TPR1 and the TPR do-termini of Hsp70 and Hsc70 as ligands (Figure 1C). A main of PP5, TPR2A contains an insertion between re-12 kDa fragment of Hsp90 (C90) that includes the dimer- peat two and three which leads to elongation of helices
B2 and A3 by one turn each (Figures 2E and 3). In bothization domain and the authentic C terminus of Hsp90
Chaperone receptors
Review: Kriechbaumer et al (2011) Protoplasma
Sgt2 is dimeric with three distinct domains
Amino terminal homodimerization domain
Binds Get5
Tobaben et al 2003
Liou et al 2005
Liou et al 2007
Central TPR domain
Binds heat shock proteins
Tobaben et al 2001
Tobaben et al 2003
Liou et al 2005
Winnefeld et al 2005
Carboxyl terminal methionine/
glutamine rich domain
Binds hydrophobic sequences
Wang et al 2003
Liou et al 2005
Wang et al 2010
Sgt2/SGTA
HumanSgt2TPR(2vyi)Dutta&Tan(2008)Biochemistry47:10123
AfSgt2 HspOrganizingProtein(HOP)
C-terminaldomain:DDVDD
HSP70peptide:DDVD-COOH
N
TPR
C
SmallHeatShockProteins
• Actasspongesforunfoldedproteins
• Familyincludesα-crystallin
• Suppressesproteinaggregation• Donotappeartohaveunfoldingandrefoldingactivity
• Highcapacityforproteinbinding:upto2denaturedproteinspersubunit
• 60%ofthelens(anavascularorgan)/90%oftheprotein
• Lenslastsforlife– Proteinlastalifetime
• Cataractsinvolveaggregationofcrystallins
• Homologsexpressedbroadly• Immunoglobulinfold• FirststructurefromM.jannaschii
α-crystallin
Kim3(1999)Nature394:595(1shs)
10.2210/rcsb_pdb/mom_2010_7
Higherorderstructure
24subunits,2,3,4foldsymmetry
Kim3(1999)Nature394:595(1shs)
Conservedfold
• Oligomerscanvaryinsizeevenwithinhomologues
• Rangefrom150kDato800kDa
Mchaourab,Godar&Stewart(2009)Biochemistry
Wheat small HSP
van Montfort et al. Nature Structural Biology (2001)Dodecameric structure
Differentstructural
organization
Haslbecketal(2005)NSMB
Functionalmodel
Haslbecketal(2005)NSMB
ΔT
Get3-likeproteinsandsHSPs
Get3 Nostocsp(3igf)
Synechocystis PCC 6803,Hohmann-Marriott et al. 2009
Hsp90Family
Taipale,Jarosz&Lindquist(2010)NatureRevMCB
Anopheles gambiaeDrosophila melanogaster
Ciona intestinalis
D. rerio (Hsp90B2)Danio rerio (Hsp90B1)
Gallus gallusHomo sapiens
Strongylocentrotus purpuratus
Caenorhabiditis elegans
Monosiga brevicollis
Arabidopsis thalianaOryza sativa
Dictyostelium discoideumPlasmodium falciparum(HSP90B1)
P. falciparum(HSP90B2)
P. falciparum
Endoplasmic reticulum HSP90(HSP90B family)
Bordetella pertussisEscherichia coli
Geobacter sulfurreducens
Wolbachia spp.
Gloeobacterviolaceus
B. fragilis(HtpG2)
S. coelicolor(HtpG2)
Borrelia burgdorferi
Helicobacter pylori
Bacteroidesfragilis
Streptomycescoelicolor
Bacterial Hsp90(HtpG family)
A. gambiae
D. rerioG. gallus
H. sapiens
C. intestinalisS. purpuratus
C. elegansD. discoideumM. brevicollis
D. melanogaster
Mitochondrial HSP90(TRAP family)
A. thaliana (HSP90-6)
A. thaliana (HSP90-7)O. sativa (HSP90C1)
O. sativa (HSP90C2)
Chloroplast HSP90(HSP90C family)
A. gambiaeD. melanogaster
C. elegans
C. intestinalis
D. rerio (Hsp90_2)D. rerio (Hsp90_1)
G. gallus(Hsp90_)
H. sapiens (HSP90_)
D. rerio (Hsp90`)G. gallus (Hsp90`)
H. sapiens (HSP90`)S. purpuratus
M. brevicollis
A. thaliana (HSP90-1)
O. sativa (HSP81-2)
A. thaliana (HSP90-2)A. thaliana (HSP90-3)
O. sativa (HSP81-3)
P. falciparumD. discoideum
Neurospora crassaS. cerevisiae (Hsc82)
Saccharomycescerevisiae (Hsp82)
O. sativa (HSP81-1)
A. thaliana (HSP90-4)
Cytoplasmic HSP90(HSP90A family)
0.1
Nature Reviews | Molecular Cell Biology
Heat shock protein(HSP). A protein induced in response to increased temperature, classified according to its size. Many HSPs function as molecular chaperones.
Co-chaperoneA protein that associates with and promotes the function of chaperones by modulating their chaperoning activity and/or regulating their substrate specificity. The definition of a co-chaperone is somewhat arbitrary, as some co-chaperones have chaperoning activity even when they are not associated with the core chaperones.
HSP90 structure. HSP90 shares structural homology with other ATPases in the GHKL (gyrase, HSP90, histidine kinase and MutL) superfamily 41. Each mono mer in the HSP90 dimer has a highly conserved
amino-terminal domain (NTD) connected to a middle domain41, followed by a carboxy-terminal domain (CTD) (FIG. 1). HSP90 binds ATP in its NTD, hydro-lysing it following the interaction with clients and certain
Box 2 | HSP90 family proteins
The heat shock protein 90 (HSP90) family is highly conserved. With the exception of archaea, organisms in all kingdoms of life have one or more genes encoding HSP90. The HSP90 family can be divided into five subfamilies: cytosolic HSP90A, endoplasmic reticulum (ER)-localized HSP90B, chloroplast HSP90C, mitochondrial TNFR-associated protein (TRAP) and bacterial high temperature protein G (HtpG)165 (see the figure; scale bar represents genetic distance). HtpG is found in most bacteria but, in contrast to its eukaryotic counterparts, it is not an essential gene in non-stressful conditions and has only modest effects on growth at high temperatures.
The four eukaryotic HSP90 subfamilies evolved from an HtpG-like group165. The TRAP family of mitochondrial HSP90s is most closely related to the bacterial HtpG group; however, it is unlikely to be of endosymbiotic origin165. Instead, it seems to have evolved independently of the HSP90A, HSP90B and HSP90C families during early eukaryote evolution. The function of TRAP is poorly understood, but human TRAP has been shown to protect cells from oxidative stress-induced apoptosis166.
The ER-localized HSP90B proteins, known as GRP94 in humans, have a role in ER protein quality control. All known GRP94 clients are in secretory pathways or are found on the cell surface167. GRP94 is strongly induced by perturbations in ER protein homeostasis, such as starvation for glucose or exposure to tunicamycin, which inhibits N-linked glycosylation of proteins. Interestingly, it seems that the cytosolic HSP90A and chloroplast HSP90C families evolved from the ER-localized HSP90B group165.
The HSP90A group is the largest, most widespread and best-studied of the HSP90 families. Evolutionarily recent duplication events have resulted in multiple genes encoding cytosolic HSP90 in many organisms. For example, most vertebrates have two genes encoding HSP90: HSP90AA/1 and HSP90AB/1. The function of these paralogues is highly overlapping, although a few paralogue-specific functions have been reported26.
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Hsp90• Can prevent
aggregation of proteins • Interact with ‘clients’
– Transcription factors and protein kinases
– Roles in signal transduction, chromatin remodeling, cellular trafficking etc…
• Late stage in folding • Can target for
degradation • Related to DNA gyrase
2iop
Hsp90insignalingandaccumulationofmutations
Calderwood..Ciocca(2006)TiBS Taipale,Jarosz&Lindquist(2010)NatureRevMCB
thus permit a time window for subsequent repair of theproteome [6,16]. The massive upregulation of HSPsthat occurs during heat shock involves facilitationof expression at every level, including activation ofthe potent heat-shock transcription factor 1 (HSF1),stabilization of HSP mRNA, selective translation ofHSPs, and stabilization of HSPs at the protein level [1,17].
As we discuss here, evolving studies indicate that thesepotent cytoprotective and folding properties of the HSPsare co-opted duringmalignant progression when theHSPsbecome expressed at high level to facilitate tumor cellgrowth and survival [18].
Essential steps in tumor progressionMost tumors are formed by a stepwise progression of cellsfrom a transformed but minimally altered state that cangrow and form nodules or polyps (in solid tumors) to amultiply deviated state that is capable of unlimitedgrowth, manipulation of the local environment, invasionof surrounding tissues and escape into the circulation tofound new colonies of secondary tumors or metastases.Such a progression involves diverse molecular andmorphological changes.
In their landmark review, Hanahan and Weinberg [19]suggested organizing these traits into six essentialalterations in cell physiology: (i) self-sufficiency in growthsignals; (ii) insensitivity to growth inhibition; (iii) evasionof PCD; (iv) limitless replicative potential; (v) sustainedangiogenesis; and (vi) tissue invasion and metastasis. Theoccurrence of this series of radical, mostly genetic,changes in physiology is thought to be at least partiallydependent on the evolving instability of the tumor genomeowing to a breakdown in the DNA repair pathways[20,21].
In addition to progressing in terms of acquiringenhanced malignant capabilities, most human tumorsundergo further selection through exposure to variousforms of cytotoxic therapy, which leads, in surviving cells,to various phenotypes that are resistant to the cytotoxictherapies being used. Increases in HSP expression seem tobe involved not only in most of these stages of tumordevelopment, but also in the acquisition of drug-resistant phenotypes.
Role of HSPs in formation of the malignant phenotypeHSP90 and self-sufficiency in growth signalsThe effects of the HSPs on the anabolic pathways leadingto the first of the essential alterations in the Hanahan andWeinberg scheme [19] – namely, self-sufficiency in growthsignals – are mediated largely by HSP90. This molecularchaperone is indispensable for stabilizing the fragilestructures of many of the receptors, protein kinases andtranscription factors that lie along the pathways of normalcellular growth [22]. HSP90 is required to maintainsignaling proteins in an active conformation that can berapidly triggered by growth signals [10,22]. Thus, HSP90might be viewed as a facilitator of the rapid and fluidresponses to extracellular signals that are requiredparticularly in development and cell renewal [22,23].Transformation involves the overexpression or mutationof many of these HSP90-dependent signaling molecules,and HSP90 is increasingly needed to maintain suchproteins in active conformation.
The extent to which HSP90 clients are essential forsignal transduction pathways that ultimately becomesubverted in cancer can be seen in Figure 1, whichshows signaling pathways emanating from the proto-oncogene HER2. HSP90 is required for the stability andactivity of HER2 itself and its downstream proteins,including the protein kinases Akt, c-Src and Raf-1,which have key roles in cell growth and survival [22].Over 100 similar HSP90 clients exist, each of which showsa similar dependency on HSP90 binding [22]. HSP90performs these molecular chaperone functions as the
Table 1. The principal HSP families in human cellsa
Protein Function Co-chaperones RefsHSP27 Molecular chaperone None [13]HSP60 Chaperonin HSP10 [11,12]HSP70 Molecular chaperone HSP40, GrpE, BAG,
HSPBP1, Hip, Hop, CHIP[2,9]
HSP90 Molecular chaperone p23, Hop, FKBP51,FKBP52, Cyp40, cdc37
[2,8,10]
HSP110 Molecular chaperone None [4]aThe co-chaperones HSP40, p23, FKBP51, 52, Cyp40 and Cdc37 facilitateinteractions with the substrate of the primary chaperone. Others, including GrpE,BAG-1 and HSPBP1, function as nucleotide exchange factors that permit efficientATP binding and hydrolysis. A third class of co-chaperones enable the molecularchaperones to interact with other proteins: Hop bridges the HSP70 and HSP90systems, and CHIP links HSP70 to the ubiquitin–proteasome system.
Ti BS
MEK
ERK
Akt
Mutation
NFκBHSF1HIF1α
Heregulin
Raf
PI3K
HER2HER3
Src
HSP90
Ras
SRF
Mutant protein
Figure 1. Role of HSP90 in intrinsic growth signaling and in the accumulation ofproteinmutations in cancer. HSP90 (blue sphere) is associatedwithmany of the keysignaling molecules that mediate autonomous growth in cancer. Shown here, forexample, are the pathways of growth and survival signaling initiated by heregulinassociation with HER3–HER2 heterodimers and (i) induction of the growth-promoting extracellular-regulated-signal kinase (ERK) pathway (yellow spheres),leading to activation of the transcription factor SRF and transcription of immediateearly genes; and (ii) the pro-survival PI3K pathway (green ovals). HSP90 bindingis required for the stability of many proteins in this pathway, including HER2, c-Src,c-Raf, ERK1 and Akt. Also shown is one of the mechanisms for HSP90 induction incancer: heregulin-induced activation of HSF1 through PI3K and Akt leads toexpression of HSP90. In addition, HSP90 is involved in a second related process: thestabilization of mutant proteins. Mutant proteins are shown binding to HSP90;arrows suggest the multiple influences of such stabilized proteins on the tumor cellphenotype.
Review TRENDS in Biochemical Sciences Vol.31 No.3 March 2006 165
www.sciencedirect.com
Nature Reviews | Molecular Cell Biology
?
?? ?
FKBPs
Nuclear receptorsSter receptors
(GR )receptors
(PP PXR)
eNOS
LRR proteins
pr ,
pr)
Transcription factorsP ,
trithor
y
ce
Ster g
HSF1
e
Prtr g
TO 0Myosin
ecce y
TERT
Te ee
Calcineurin
g
Viral proteins
pic vir psidprreopr
Kinases
(v-Sr
C
Mitochondrial proteins
?
?
HSP90
telomerase reverse transcriptase (TERT)106, and trans-cription factors and chromatin proteins (such as PAS family transcription factors, p53, STAT3 and tri-thorax)107–110 (FIG. 3). Collectively, these place HSP90 at numerous vital regulatory hubs.
In plants, HSP90 and the co-chaperone SGT1 asso-ciate with many NLR (nucleotide-binding, Leu-rich repeat (LRR)) proteins, which are cytoplasmic sensor proteins required for innate immunity111,112. Their LRR domains recognize pathogen effector molec-ules, which leads to the activation of downstream
signalling. HSP90 and SGT1 are required for the stability of the sensor complex111,112, so mutations in HSP90 or SGT1, or inhibition of HSP90, reduce the levels of NLR proteins and compromise the immune response against many pathogens111,112. Interestingly, mammalian NOD-like receptors, which are structur-ally related to plant NLRs, also functionally interact with HSP90 and SGT1 (REF. 113). As HSP90 has also been implicated in antigen processing114,115, it seems it has a role in both innate and adaptive immunity in vertebrates.
Figure 3 | HSP90 regulates diverse cellular processes through its interaction with client proteins. Heat shock protein 90 (HSP90) associates with core co-chaperones (such as p23 (also known as PTGES3 and as Sba1 in yeast), HSC70 and HSP90-organizing protein (HOP; also known as p60 and STI1) and the HSP40–HSP70 chaperone system; orange) and other co-chaperones (green). Adaptor co-chaperones such as Cdc37 and SGT1 associate with a limited number of proteins or protein domains (blue). However, for most HSP90 clients, it is not known which co-chaperones, if any, are associated in the complex (indicated by question marks). AHR, aryl hydrocarbon receptor; AR, androgen receptor; CDK4, cyclin-dependent kinase 4; EGFR, epidermal growth factor receptor; eNOS, endothelial nitric oxide synthase; ERα, estrogen receptor-α; FKBP, FK506-binding protein; GR, glucocorticoid receptor; HAP1, haeme activator protein 1; HCK, haemotopoietic cell kinase; HCV, hepatitis C virus; HIF, hypoxia-inducible factor; HSF1, heat shock factor 1; LRR, Leu-rich repeat; MR, mineralocorticoid receptor; N, nucleoprotein; NALP, NACHT, LRR and PYD domains-containing protein; NLR, nucleotide-binding LRR; NOD1, nucleotide-binding oligomerization domain-containing protein 1; PPARα, peroxisome proliferator-activated receptor-α; PR, progesterone receptor; PRF, Pseudomonas resistance and fenthion sensitivity; PXR, pregnane X receptor; RPS2, resistance to Pseudomonas syringae 2; STAT3, signal transducer and activator of transcription 3; TERT, telomerase reverse transcriptase; TOM70, translocase of outer membrane 70 kDa.
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Lot’s of structures in different conformations from a variety of methods. General path toward unified model.
Mayer (2010) Mol Cell
Hsp90 Cycle
Substratemodel?
Street,Lavery&Agard(2011)MolCell
OverallCycle
Taipale,Jarosz&Lindquist(2010)NatureRevMCB
HSP40
Nature Reviews | Molecular Cell Biology
ADPADP
ATP ATP p23p23
CYP40 CYP40
Client
Client
ATP
HSP70
HSP40 ClientHSP70
HOP
HOP
Mature client
Chaperone cycle
a
NTD
CS
CC CTD
NTD CTD AHA1
TPR CS SGS
Cdc37
SGT1
p23
Bind HSP90 N-terminal domain
g
TPR TPR
FKBP TPR
PI TPR
TPR TPR
TPR HOP
TPR
TPR
U-box CHIP
WISp39
FKBP52 and FKBP54
CYP40
PP5
TOM70
TPR2
Bind HSP90 C-terminal MEEVD
Binds HSP90 middle domain
TPR Phosphatase
TPR
TPR TPR DnajC-terminal MEEVD
C-terminal MEEVD
c Cdc37b d p23
e AHA1 f SGT1
HSP90’s middle domain catalytic loop and the NTD nucleotide-binding site. This stabilizes an HSP90 con-formation that promotes efficient ATP hydrolysis (FIG. 2f). Indeed, recent studies suggest that AHA1 stimulates the ATPase activity of HSP90 through an asymmetric interaction that promotes a closed conformation in which the NTDs from each HSP90 monomer associate81.
Interestingly, the ability of AHA1 to accelerate the rate limiting conformational changes that precede ATP hydrolysis can be seen even in the absence of bound nucleotide63.
Two structures illustrate how co-chaperones inhibit HSP90 activity. Cdc37 links kinase clients to HSP90, interacting with the kinase through its N terminus and
Figure 2 | Co-chaperones facilitate client maturation by interacting with distinct surfaces on HSP90. a | Schematic of the maturation of the progesterone receptor mediated by the cooperation between heat shock protein 90 (HSP90), HSP70 and co-chaperones. The progesterone receptor binds HSP40 and HSP70, which then binds the HSP90–HOP (HSC70 and HSP90-organizing protein; also known as p60 and STI1) complex, leading to delivery of the client. Finally, the co-chaperone p23 (also known as PTGES3 and as Sba1 in yeast) and ATP bind the complex, promoting client maturation and complex dissociation. The role of cyclophilin 40 (CYP40; also known as PPID) is not well understood but it regulates the activity of client proteins in the final stages of the chaperone cycle. b | The crystal structure of yeast Hsp90 in a closed conformation, bound to a non-hydrolysable ATP analogue (AMPPNP) and the co-chaperone Sba1. Co-chaperones can bind the Hsp90 amino-terminal domain (NTD; orange), middle domain (green) or the Met-Glu-Glu-Val-Asp (MEEVD) motif in the carboxy-terminal domain (CTD; blue). c–f | Cdc37 and p23, which both inhibit HSP90 ATPase activity, bind near the ATP-binding pocket of HSP90. The co-chaperone activator of HSP90 ATPase homologue 1 (AHA1) binds along the HSP90 middle domain and facilitates ATP hydrolysis by promoting middle domain contacts with the active site in the NTD. SGT1, which, like p23, binds HSP90 through its CHORD and Sgt1 (CS) domain, interacts with a distinct site on the HSP90 NTD. g | Domain organization of co-chaperones that have been shown to bind HSP90. Domains that interact with HSP90 are coloured as in parts c–f. CC, coiled coil; CHIP, C terminus of HSP70-interacting protein; FKBP, FK506-binding protein; PI, prolyl isomerase; PP5, protein phosphatase 5; SGS, SGT1 specific; TOM70, translocase of outer membrane 70 kDa; TPR, tetratricopeptide repeat; WISp39, WAF1/CIP1 stabilizing protein 39.
REVIEWS
520 | JULY 2010 | VOLUME 11 www.nature.com/reviews/molcellbio
© 20 Macmillan Publishers Limited. All rights reserved10
Overlapinbinding Co-chapabundance
Mayer&LeBreton(2015)MolCell
Asymmetryessential?
Mayer&LeBreton(2015)MolCell
Hsp90andEnvironmentalStressTransformtheAdaptiveValueofNaturalGeneticVariation
Jarosz&Lindquist(2010)Science
AAAATPasefamily• ATPasesAssociatedwithvariouscellularActivities
– Diversefamily– Unfolding/unwinding,assembly/disassembly
– Oftentransitionbetweenmonomersandoligomers
• Hsp100/Clpfamily– chaperones/proteases,
– componentsinDNAreplication,recombinationandrestriction,theNSFproteininvesiclefusion,dyneinmotorproteinsandmanyothers
• Hsp100proteinscantotallyunfoldtheirsubstrateproteins–deliverto:– Proteases– Hsp70Pathway
ClpXstructures
Mayer(2010)MolCell
Hsp100ATPcycle
Mayer(2010)MolCell
Chaperone Summary
Review: Hartl (2002) Science 295:1852 Kim..Hartl (2013) Ann Rev Biochem 82:323
Prefoldin, a functional substitute for Hsp70 chaperones
• Minimal chaperone machinery in bacteria – nascent chain binding chaperones (trigger factor and
Hsp70)– Group I chaperonin system GroEL/GroES (acts post-
translationally)• Archaea have thermosome (double ring structure like
GroEL/GroES) but most lack Hsp70 equivalent • Instead of Hsp70, Archaea use prefoldin (absent in
bacteria but found in eukaryotes) to stabilize non-native proteins for subsequent folding in the cavity of a chaperonin
Hartl lab: Siegert et al Cell (2000) 103: 621-32 (1fxk)
• Found in eukaryotes and archae • Can substitute for Hsp70 • 2 proteins (ratio 2α :4β) • Hexameric complex (jellyfish?) • Six tentacles (rods of - α helical coiled
coils) pointing away from two 8-stranded anti-parallel β-barrels
Structure of prefoldin
α
ββββ
β
αα
Prefoldin
Hartl lab: Siegert et al Cell (2000) 103: 621-32 (1fxk)
• Hydrophobic patches on distal regions point to central cavity
• Flexible tentacles – to adjust to different sized substrates– nascent chains and partially folded proteins
• Multivalent binding to substrate – detergent-like solubilization of non-native
polypeptides prone to aggregation• Substrates may protrude into central cavity
– shields aggregation-prone states from bulk solution
• Substrate binding may cause unwinding of coiled coils, exposing more hydrophobic regions
• Binding to individual tentacles – may be weak and reversible– allows a strong interaction when all six are used – can release to downstream chaperones without
ATP hydrolysis.
Skp, a related protein
Found in the periplasm 1u2m and 1sg2
Walton & Sousa (2004) Mol Cell 15:367-74Korndorfer et al. (2004) NSMB 11:1015
Trigger factor
Banlab:FerbitzetalNature(2004)431:590-6(1w26&1w2b)
• Trigger factor found in all eubacteria• Member of a group of ribosome associated chaperones• Binds to 50S subunit at exit tunnel• Helps nascent proteins fold
Hydrophobic Hydrophilic
Trigger factor mechanism
• Trigger factor waits at exit tunnel
• Cavity facilitates folding • When the nascent domain is
big enough TF falls off
Banlab:FerbitzetalNature(2004)431:590-6
Xu etal Nature Struct Biol (2000) 7:1172-7 (1fx3)
SecB
SecA
Modified from Wickner & Schekman Science (2005) 310:1452-6
Bacterialpost-translationaltranslocation
• SecB stabilizes unfolded state for delivery to membrane
• SecA causes release
Proteinmo=fs
• SecondaryStructure
• Mo=f–smallestfoldingstructure
• Domain(definestopology)–foldingregion
• Monomer
• Oligomer
Modularsmalldomains
• Proteinsinvolvedincellregula=on• Interac=ondomains-recogni=onmodules
– Targetanac=vitytoasubcellularloca=on– Independentlyfoldedmodules
• Bindpartnersalone• Closeaminotermini-bindingonoppositesurface
– Familiesdividedbysequence,structureandligand• Proline-rich(SH3,WW&EVH1)• Phosphotyrosine(SHT&PTB)• Phosphoserine/threonine(FHA,PDB,WD40)• Phospholipids(PH&FYVE)
– Cancombineinanyorderforspecificfunc=on
PetskoG.A.,Ringe,D.,ProteinStructureandFunc>on2004,figure3-2(upper),pg.89.
Interac=onDomains
PetskoG.A.,Ringe,D.,ProteinStructureandFunc>on2004,figure3-2(middle),pg.89.
Interac=onDomains
Petsko G.A., Ringe, D., Protein Structure and Function 2004, figure 3-2 (lower), pg. 89.
Interac=onDomains
WD40inac=on• RACK1-ascaffoldprotein• Interactssimultaneouslywithseveralsignaling
molecules• Linkstransla=ontothesignaltransduc=on
pathway
SenguptaetalNSMB(2004)11:957-62NissonetalEMBOreports(2004)5(12)1137-41
Mosaicproteins• Ig–immunoglobulindomain• Fn1–fibronec=ntypeI• Kringle–bindsamediator• Fz–frizzledCRDdomain–binds
Wntpathwayproteins• EGFdomain–proteinbinding
domain
Tissueplasminogenac=vator
Receptortyrosinekinase
Fig.2.27Fromthetext
Kringle-1tpkKossiakofflab
Fz–1ijyLeahylab
EGFdomain–1apo Fn1domain(2rky)
Igdomain(1aqk)FromFig.2.31
Proteinkinases
Fig.11.4(1hck&1fin)
Twodomainsinanac=veandinac=veform(cyclin-dependentkinase)
Solublekinases
Fig.11.5,11.6&11.8
SH2domain(1sps)
Inac=veSrctyrosinekinase
• Verymodular• Dimeriza=onleadsto
ac=va=on• Raspathway
Fig.11.10,11.11&11.12
Membranereceptors
Growthofproteintopologies
• PDBgrowthcon=nuestorise• GrowthofnewtopologiesdoesnotcorrelatetoPDBGrowth
PDBstatsfromhop://www.rcsb.org/
Foldsinproteinstructure
• ClassArchitectureTopologyHomologousSuperfamily– Orengo,Michie,Jones,Jones,Swindells,andThornton(1997)“CATH-AHierarchicClassifica=on
ofProteinDomainStructures”Structure5:1093– hop://www.cathdb.info/
• StructuralClassifica=onofProteins– Murzin,Brenner,Hubbard,Chothia(1995)“SCOP:aStructuralClassifica=onofProteinsdatabase
fortheinves=ga=onofsequencesandstructures”JMolBiol247:536– hop://scop.berkeley.edu/
• DistanceMatrixAlignment– HolmL,KaariainenS,RosenstromP,SchenkelA.(2008)“Searchingproteinstructuredatabases
withDaliLitev.3”Bioinforma=cs2008
• SimpleModularArchitectureResearchTool– Canlookupdifferentdomainstoseehowthey’rearrangedinproteinsandcompareproteins
fromdifferentgenomes– Schultzetal(1998)“SMART,asimplemodulararchitectureresearchtool:Iden=fica=onof
signalingdomains”PNAS95:5857
Brenner et al COSB (1997) 7:369
SCOPStructural Classification Of Proteins
hop://scop.berkeley.edu/
DALIDistance matrix ALIgnment - fold similarities
Holm & Sanders Science (1996) 273:595http://www.ebi.ac.uk/dali/
Func=oncanevolveseparately
Chymotrypsin Sub=lisin
Fig.2.30fromthetext
Twodifferentserineproteases
Whyareproteinssobig?Whydocellsbuildoligomericproteins?
• Minimumsize– Stability(smallproteinshavemoredisulfidesormetals)– Smallproteinshavelargesubstrates(DNAbinding)
• Monomers:manysecretedproteins(e.g.,toxins,hormones)• Oligomers:manyintracellularproteins
– averageoligomericstateinE.coliis4• Smallmonomerscandiffusetointeractwithlargesubstrates
– e.g.,electrontransportproteinsthatinteractwithimmobilesubstratesinlipidbilayer
• Enzymesthatactonsmallsubstratescanbelarge-onlylimitedbydiffusion
Goodsell&Olson(1993)Solubleproteins:size,shape,&func=on.TIBS18:65-68
Sampleofsolubleproteinsdrawntorela=vescale
Toxins
Electrontransferproteins
Hormones Carrierproteins
Goodsell&Olson(1993)TIBS18:65-68
WeirdShapeHydrolase
Sampleofenzymesdrawnatrela=ve
scale
Goodsell&Olson(1993)TIBS18:65-68
Enzymes100-750aa
Examplesofoligomericproteins
Homo-oligomers:• Alcoholdehydrogenase(1HTB):2• Phosphofructokinase(4PFK):4• Glutaminesynthetase(1FPY):12
Hetero-oligomers• Hemoglobin(2DHB):4-α2β2• Aspartatetranscarbamylase(1AT1):12-C6R6• Protease/proteaseinhibitors(non-obligate
oligomers)
PetskoG.A.,Ringe,D.,ProteinStructureandFunc>on2004,figure1-74(upper),pg.45.
Oligomers
D-aminoacidaminotransferase3DAA
KDGPaldolase1FQ0
Neuramidase1A4Q
Lactatedehydrogenase
1LDN
Choleratoxin1CHP
Insulin4INS
Molybdenumcofactor
biosynthesisproteinC1EKR
GroESco-chaperonin1G31
Galactonate
dehydratase2GSH
3-dehydroquinatedehydratase2DHQ
Rhinovirus1AYM
Proteasome1G65
Advantagesofquaternarystructure
• Coopera=vityandsubstratefeedback– ac=vesiteouenlocatedatsubunitinterface
• Evolu=on– Muta=onsareamplified– Badmuta=onsarehardtointroduce
• Gene=ceconomy– Savestheneedforlongtranscripts
• Reducestheerrorrateofsynthesis– If1errorper1000residues:#correctcopies– Singlechain(1000residues) 37%– Fourchains(250residueseach) 78%
• Reducesurface/volume– buriespor=onsofproteinsurface– reduce#ionsneededtoneutralizeexposedcharges– reduce#orderedwatersforhydra=on
Goodsell&Olson(1993)Solubleproteins:size,shape,&func=on.TIBS18:65-68
Protein-ProteinInterfaces
• InterfaceASAlinearlyrelatedtoMW(r=0.69)
• ASAburied(persubunit)varies368.1to4746.1Å2
• ASA/MWdecreasesfromprotomertodimer
• Hydrophobicityattheinterface:– exterior>interface>interior– Atoms:68.1%non-polar– Residues:46.7%hydrophobic,31.4%
polar,21.9%charged• Interfacehasaffinityforhydrophobic
residues• Average0.88H-bondsper100Å2
Jones&ThortonProgBiophysMolBiol(1995)63:31
Predic=ngdimerinterfaceBsdimer5442Å2 Bsmono2410Å2
Mt2822Å2
TheSecAATPaseisknowntohaveadimericform;however,inthreecrystalformstherearethreedifferentinterfaces!
Whichisthephysiologicaldimer?
OsborneetalPNAS(2004)101(30):10937-42
Can’talwayspredict
• Twohomologoushomodimers
• Twodifferentpacking• 1ALO&1FIQ
Valdar&ThorntonJMB2001(2)399
Commonmodifica=ons
• Hydrophobicaddi=on– N-terminal
• Myristoyla=on(membraneanchoring)
– C-terminal• Prenyla=on(membraneanchoring)
• GPIanchor(targe=ng)
• Glycosyla=on– TransferofasugartoAsnorThr/Ser
• Phosphoryla=on– Centralplayerinsignalling– Reversible– Kinases
• Morethan700proteins• Commonfold(~250residues)• TypicallyTyrandSer/Thr
– Phosphatases• Proteinserine-threoninephosphatases
• Proteintyrosinephosphatases(PTP)
• HC(X)5Rmo=f
Rarermodifica=ons
• Hydroxyla=on– Remembercollagen(hydroxyproline)
• Oxida=on– Duetoreac=veoxygen
– Typicallybad(exceptincysdrivenredoxreac=ons)
• Deamida=on– SpontaneousconversionofAsn&Glntoacid
SHC
HOSC
HOO S
C
CH3
S
CC
O
CH3
S
CC
O O
CH3
S
CC
Cy steine
Methionine
N H3+
C
C
C
C
H OC
C
C
C
Ly sine
C
NH
N
O
C
NH
NH
Histidine
NH2+NH2C
N
C
C
C
OH
C
C
C
Arginine
O
NH2
O
NH
NH
R1
O
OH
O
NH
NH
R1
O
O
NH
N
R1
O
OHCC
O
NH
NH
R1
NH
C OH
O
O
C NH
R1
Succinimide
Asn-R1
Asp-R1
Asp-R1 is oAsp-R1
NH2 H2O
OHOO S
C
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