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