Proc. Nadl. Acad. Sci. USAVol. 91, pp. 11197-11201, November 1994Biochemistry

The ability of the immunophilin FKBP59-HBI to interact with the90-kDa heat shock protein is encoded by its tetratricopeptiderepeat domainCHRISTINE RADANYI*, BEATRICE CHAMBRAUD*, AND ETIENNE-EMILE BAULIEUtInstitut National de la Santd et de la Recherche MWdicale (U33), and Colifge de France, 80, rue du G6ndral Leclerc, 94276 Bicetre Cedex, France

Contributed by Etienne-Emile Baulieu, July 28, 1994

ABSTRACT A protein of apparent molecular mass of =59kDa of the FK506-binding protein class (FKBP59) has beenfound associated with the heat shock protein hsp90 included innontransformed steroid receptor complexes and termedFKBP59-HBI (HBI for Heat shock protein 90 Binding Immu-nophilin). Further data analysis has revealed that this immu-nophilin also belongs to the tetratricopeptide repeat family ofproteins. In this work, we describe the hsp90-binding domainof FKBP59-HBI. Density gradient centrifugation, gel ifitra-tion, and immunoadsorption analyses failed to demonstrate astable association between FKBP59-HBI and hsp90 in therabbit reticulocyte lysate. Using a gel-retardation assay, weprovide evidence for a specific ATP-independent interactionbetween highly purified wild-type rabbit FKBP59-HBI andhuman hsp9OI3. This interaction was not affected by theimmunosuppressants FK506 and rapamycin. Examination ofthe behavior of several mutants led us to conclude that thetetratricopeptide motifs localized in the C-terminal part ofFKBP59-HBI are necessary for hsp90 binding.

binding domains has permitted investigation of some func-tions of the protein (13). It has been confirmed that thepeptidyl-prolyl isomerase activity and the binding of immu-nosuppressants are confined to the N-terminal domain (13,14). In spite of the enzymatic and immunosuppressant-binding activities of FKBP12 and FKBP59-HBI, the latter,even bound to FK506, does not show any association with,or inhibition of, the phosphatase activity ofcalcineurin (6, 15,16) as do the FKBP12-FK506 complexes. The FKBP59-HBIII domain is able to bind ATP/GTP (17). The binding ofhsp90to FKBP59-HBI has not yet been studied in detail and definedtopologically. The first aim of this paper is to report on thedemonstration of the complex formation between purified,both wild-type and mutated, rabbit FKBP59-HBI and humanhsp90 overexpressed in Escherichia coli. We also presentevidence that the ability of FKBP59-HBI to interact withhsp90 is encoded by the TPR domain of the protein, asequence that has been recently identified in many importantproteins dealing with cell division in yeast and other orga-nisms (18, 19).

A protein of apparent molecular mass of -59 kDa (p59) wasoriginally identified as a component of unliganded, nontrans-formed steroid hormone receptor oligomeric complexes (1,2), which also include the heat shock protein hsp90 (3).Binding of this protein to hsp90 and not to the hormone- andDNA-binding receptor unit has been reported in vitro (2) andin vivo (4). Recently, elucidation of the primary structure ofthe rabbit p59 (5), confirmed by that of its human equivalent(6), sequence alignment, hydrophobic cluster analysis of thesecondary/tertiary structure (7), and biochemical data (par-tial amino acid sequence, peptidyl-prolyl isomerase activity,and binding of the immunosuppressants FK506 and rapa-mycin) (8, 9), has led to the identification of p59 as animmunophilin of the FK506-binding protein (FKBP) class.We therefore referred to it as FKBP59-HBI (HBI for Heatshock protein 90 Binding Immunophilin). Hydrophobic clus-ter analysis has suggested that there are three domainsstructurally related to the well-conserved FK506-bindingprotein of 12 kDa (FKBP12), particularly the N-terminaldomain with 49% homology (7). A putative calmodulin-binding site located at the C-terminal extremity of the proteinhas been detected (7), and indeed binding of calmodulin toFKBP59-HBI has been reported (10). Moreover, data baseanalysis revealed that FKBP59-HBI is also a member of thetetratricopeptide repeat (TPR) family of proteins, and, inparticular, it shares "30% homology with a similar TPRfound in cyclophilin 40, a cyclosporin A-binding protein of'"'40 kDa, found as a component of nontransformed estrogenreceptor (11, 12).Recombinant full-length FKBP59-HBI and a series of

mutants delimiting the three FKBP-like and calmodulin-

MATERIALS AND METHODSMaterials. ECi is a monoclonal antibody against FKBP59-

HBI (20). The polyclonal antipeptide antibody 173 againstFKBP59-HBI has been described (5), and the polyclonalantipeptide antibody 174 raised against the C-terminal part ofhsp90 was obtained from Neosystem (Strasbourg, France).Species-specific monoclonal antibodies against chickenhsp90 (BF4) and rabbit hsp90 (7C10) have been reported (3,21). N27, a monoclonal antibody specific for hsp70, was fromStressGen Biotechnologies (Sidney, Canada).

Expression Vectors. All FKBP59-HBI and mutant expres-sion vectors except those mentioned below have been de-scribed (13). The full-length cDNA of FKBP59-HBI wasexcised from the vector pGEM-7Zf(+) (Promega) usingEcoRI and then inserted into the EcoRI site ofthe pGEX-1ATvector (Pharmacia LKB) to yield HBI WT. HBI 1+11+IIIBamH1 was prepared by cleaving HBI WT at the uniqueBamHI site in the open reading frame (located at amino acid400) and insertion at the BamHI site of the pGEX-1ATpolylinker. HBI I+II+IV was generated by deletion of theEco47III-Bal I fragment from the mutated F-KBP59-HBI,called HBI M3 here, cloned into pGEM-7Zf(+) (13) andinsertion into the EcoRI site of the pGEX-1AT vector. ThepKN1-3 plasmid, which contains the entire human hsp90(3sequence, was first cleaved by SnaBI, which cut 22 bpupstream of the hsp90 coding sequence, followed by Sal Ihydrolysis. This fragment was inserted into the EcoRI re-striction site ofthe pGEX-1AT vector, leading to recombinanthsp90 used for overexpression in E. coli. For translation

Abbreviations: TPR, tetratricopeptide repeat; FKBP, FK506-binding protein.*C.R. and B.C. contributed equally to this work.tTo whom reprint requests should be addressed.

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11198 Biochemistry: Radanyi et al.

experiments, the pKN1-3 plasmid was cleaved by Sal I, andthe fragment was inserted into the Xho I site of the pGEM-7Zf(+) vector.In Vitro Transcription and Translation. Translation reac-

tions in nuclease-treated rabbit reticulocyte lysate (50 "Id)were performed as recommended by the supplier (Promega).

Density Gradient Ultracentrifugation. Sedimentation anal-yses were performed on 5-20% sucrose gradients in TGbuffer [20 mM Tris-HCI/10% (vol/vol) glycerol, pH 7.4] in aVti8O rotor (Beckman) for 2 hr at 70,000 rpm or on 10-35%glycerol gradients in an SW60 rotor (Beckman) for 16 hr at48,000 rpm at 40C as described (22).

Gel Filtration. Analytical filtration on Ultrogel AcA34 wascarried out at 40C as reported (23).

Electrophoresis, Autoradiography, and Immunoblotting.Aliquots of gradient or gel-filtration fractions were submittedto SDS/7.5% PAGE and visualized by fluorography. Forimmunoblotting, the proteins were transferred to Immobilonmembranes (Millipore) and processed as described (21). Thestaining of the antigen-antibody complexes was performedwith the Vectastain ABC kit (Vector Laboratories).

Overexpression of FKBP59-HBI and hsp9O. Affinity puri-fication of full-length and truncated FKBP59-HBI and hsp9owas achieved as described (13, 24). Briefly, fusion proteinsbound to glutathione-Sepharose beads were cleaved over-night at 40C with 6 units of human thrombin (specific activityof2890 units/mg; Sigma) and dialyzed against TG buffer, andprotein concentrations were measured as reported (25), usingbovine serum albumin as standard.Nondenaturing Gel Electrophoresis. Protein samples were

run on 7.5% minislab gels as described (26). Gels were eitherstained with Coomassie blue R250, or proteins were trans-ferred to Immobilon membranes.

Immunoaffinity Chromatography. After translation, sam-ples were applied to specific (7C10) or control (BF4) anti-hsp90 immunoadsorbent prepared and processed as de-scribed (21, 23). The eluates were subjected to SDS/PAGEand immunoblotting with 7C10 and 173 followed by fluorog-raphy.

RESULTSSedimentation Analysis of in Vitro-Translated and Endoge-

nous FKBP59-HBI. To study FKBP59-HBI-hsp90 com-plexes, our general approach was to use the rabbit reticulo-cyte lysate as an expression system and a specific monoclo-nal antibody (7C10) raised against rabbit hsp90 (21). Thetranslation product ofFKBP59-HBI mRNA was analyzed bysucrose density gradient ultracentrifugation followed bySDS/PAGE and autoradiography. As shown on Fig. 1A, apredominant band with an apparent molecular mass of59 kDasedimented at 3.5-4 S. In the presence of high salt (0.3 MKCl), the sedimentation coefficient of FKBP59-HBI re-mained unchanged (data not shown). The monoclonal anti-body 7C10 was unable to shift this 3.5-4S entity, in contrastto the 6.5S dimeric form of endogenous hsp9O of the reticu-locyte Iysate, which was displaced to -9 S (data not shown).The results indicate that, under these experimental condi-tions, in vitro-translated FKBP59-HBI is not associated withhsp90. Similar results were obtained when ultracentrifuga-tion was performed for 2 hr, thus suggesting that FKBP59-HBI-hsp90 complexes had not dissociated during the courseof the experiment. However, one must be cautious since thesamples were subjected to high centrifugal forces, whichcould disrupt weak interactions. Because several laborato-ries reported the reconstitution of steroid hormone receptorsincluding hsp9o, hsp70, and the equivalent of FKBP59-HBIin the rabbit reticulocyte lysate (27-30), we examined the sizeof endogenous FKBP59-HBI. Fig. 1B shows a Western blotof an untranslated reticulocyte lysate after density gradient

AkDa708-

y

4. - Belq

40 .043.6--

BkDa105-

4WUW "PI

708-

4366-

FIG. 1. Density gradient analysis of FKBP59-HBI and hsp90. Invitro-translated FKBP59-HBI (A) and untranslated rabbit reticulo-cyte lysate (B) were layered on the top ofa 10-35% glycerol gradientin TG buffer and run for 16 hr at 48,000 rpm. Standards were glucoseoxidase (GO, 7.9 S) and peroxidase (P, 3.6 S). After SDS/7.5%PAGE, 35S-labeled FKBP59-HBI was detected by fluorography (A)and FKBP59-HBI and hsp90 were detected by Western blotting (B)with 173 and 7C10, respectively. The molecular size markers (BRL)were phosphorylase B (105 kDa), bovine serum albumin (70.8 kDa),and ovalbumin (43.6 kDa).

ultracentrifugation and SDS/PAGE. This experiment clearlydemonstrates that although some FKBP59-HBI cosedi-mented with the 6.5S dimeric form of hsp90, most wasrecovered in the 4S region of the gradient. Again, no shift ofFKBP59-HBI occurred upon incubation with 7C10. Despitethese controversial results, we performed another set ofexperiments to detect FKBP59-HBI-hsp9O complexes.Translation of FKBP59-HBI in the presence of an additionalATP-generating system, cotranslation with either the humanestrogen receptor or hsp90, and cross-linking attempts witha reversible reagent failed to demonstrate a specific interac-tion between FKBP59-HBI and hsp90. We also hoped thatwe might be able to study this interaction by performingcotranslation of both proteins in the wheat germ lysate,where no endogenous hsp90 has yet been identified. How-ever, while hsp90 migrated at 6.5 S, FKBP59-HBI wasrecovered in the first fractions of the gradient, suggestingaggregation of the protein.Lack of Demonstration of FKBP59-HBI and bsp9O Com-

plexes by Gel-Filtration Analysis and Imi n Chro-matography. The association between FKBP59-HBI andhsp90 in the rabbit reticulocyte lysate was also examined bygel filtration. As shown in Fig. 2, we observed neithercoelution of in vitro-translated (R. = 4.5 nm) nor endogenousFKBP59-HBI (same value) with hsp90 (R, = 6 nm). How-ever, even if FKBP59-HBI was present in untreated lysatecoeluted with hsp90, as reported recently (31), no shiftoccurred in the presence of 7C10 (data not shown). The datapresented above demonstrate that, in the rabbit reticulocytelysate, FKBP59-HBI is not associated with hsp90, althoughwe cannot completely rule out that our experimental proce-dures would cause dissociation of unstable complexes.Therefore we tried to immunopurify translated FKBPS9-HBIon 7C10 immunoadsorbent. After gentle washing and elution,unlike hsp90, which was specifically retained by 7C10 and notby the control anti chicken hsp90 antibody BF4, in vitro-translated FKBP59-HBI was present in both eluates, sug-gesting nonspecific interaction with the immunoaffinity ma-trix (data not shown).

Proc. Natl. Acad. Sci. USA 91 (1994)

Proc. Natl. Acad. Sci. USA 91 (1994) 11199

AkDa70.8 -

436-

1 2 3B j i ikDa105- -

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4

708-

43.6-

FIG. 2. Gel filtration analysis ofFKBP59-HBI, hsp9O, and hsp7O.In vitro-translated FKBP59-HBI (A) and untranslated rabbit reticu-locyte lysate (B) were applied to an Ultrogel AcA34 column equili-brated in TG buffer. The arrows indicate thyroglobulin (1; Rs = 8.6nm), ferritin (2; R, = 6.1 am), alcohol dehydrogenase (3; Rs = 4.5nm), and hemoglobin (4; R, = 3.1 nm). After SDS/7.5% PAGE,25S-labeled FKBP59-HBI was detected by fluorography (A), andFKBP59-HBI, hsp7O, and hsp9O were revealed by Western blotting(B) with I73, N27, and 710, respectively.

Detection of FKBP59-HBI-hsp9O Complexes by Nondena-turing Gel Electrophoresis. After their expression in E. coli,we then investigated the hsp9O-binding domain on FKBP59-HBI, schematically represented in Fig. 3, by first testing theability of full-length wild-type FKBP59-HBI (HBI WT) tostably associate with hsp9O in nondenaturing PAGE. Inpreliminary experiments, upon incubation of highly purified

I

HBI WT NH21

I6

HBI M3

IHBI I+I+ 3r Bam H1 -

HBI WT with hsp9O, a new protein band of slower electro-phoretic mobility was detected after Coomassie blue stainingof the gel. As a negative control, parallel incubation withhuman transferrin did not modify the migration of bothproteins, thus suggesting that the retarded band corre-sponded to specific HBI WT-hsp9O complexes. This newband of protein was recognized by both FKBP59-HBI andhsp9O antibodies (Fig. 4), thus confirming the associationbetween the two proteins. Moreover, addition of increasingamounts of hsp9O resulted in the total disappearance of thesignal corresponding to HBI WT with the concomitant ap-pearance of the slower migrating immunoreactive band (Fig.4C). It must be mentioned that, since hsp9O does not migrateas a single entity on SDS/PAGE where degradation productscan be visualized, the concentration of hsp9O able to interactwith FKBP59-HBI is difficult to estimate and, therefore, theexact stoichiometry of the complexes cannot be determinedaccurately. Nevertheless, these observations clearly demon-strate that purified recombinant FKBP59-HBI and hsp9O areable to associate in an ATP-independent way and form stablecomplexes that can be identified and characterized by gel-retardation assay. Therefore a more detailed analysis of theFKBP59-HBI domain involved in this interaction was under-taken.Mapping of FKBP59-HBI hsp9O-Binding Domain. The dif-

ferent constructs used for this analysis are illustrated sche-matically in Fig. 3. As shown in Fig. 5, no specific immuno-reactive bands corresponding to HBI I+II and HBI I+II+IVwere shifted upon incubation with hsp9O (Fig. 5, lanes 1-4),thus strongly suggesting that domain III is necessary, butperhaps not sufficient, to associate or maintain a structureable to bind hsp9O. Indeed, we observed that HBI I+II+IIIalso failed to interact with hsp9O (Fig. 5, lanes 5 and 6) as thefull-length mutated HBI M3 (data not shown). Given theresults obtained with the HBI WT and recently found struc-tural features of the protein-namely, identification of TPRmotifs located at the C-terminal part schematically repre-sented in Fig. 3-we first speculated that delineation of the

Z I273 306 321 355 389

1 1~IIIITPR1 TPR2 TPR3

I 11148 267 374

1 1-

400-__ IlI -iI I~~~~~~~~~~~~~~~~~~~~

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458

IHBI I+I+J= I +

IHBI I+I+=

HBI I+

374

I -

11 267

I I267

1 1~~~~~~~~~~~~~~~~~~~~~~

FIG. 3. Construction of FKBP59-HBI mutants. The structure of FKBP59-HBI cDNA is shown schematically; the numbers refer to aminoacid positions. TPR domains are represented by black boxes.

374 I 458

Biochemistry: Radanyi et al.

1 2 3 4

i f f i

11200 Biochemistry: Radanyi et al.

A BFKBP59-HBI + + + - + -

HSP 90 - - + + + +

Transferrin + - - + - -

C

HSP90 - lx 2x 3x 4x

FIG. 4. Analysis ofFKBP59-HBI-hsp90 complexes. (A) HBIWT(9.5 pmol), alone or incubated with transferrin (33 pmol) or hsp90 (33pmol) as indicated, was subjected to nondenaturing PAGE followedby Western blot analysis using the monoclonal antibody ECL. (B)hsp90 (33 pmol), alone or incubated with transferrin (8 pmol) or HBIWT (8 pmol) as indicated, was subjected to nondenaturing PAGEfollowed by Western blotting analysis using the polyclonal antibody174. (C) HBI WT (8.4 pmol), alone or incubated with increasingamounts of hsp9O as indicated, was analyzed and revealed as in A.lx-4x indicate a 1- to 4-fold excess of hsp90.

third and fourth domains (7) might not be accurate forstudying FKBP59-HBI-hsp90 interactions. Second, in lightof these observations, we must also emphasize that the twoamino acids introduced between domains III and IV of HBIM3 are located in the second helix, called the B domain ofthethird TPR motif. Given the importance of these motifs inprotein-protein interactions, as discussed below, it seemedlikely that introduction of this mutation could modify thethree-dimensional structure of at least the C-terminal part ofthe molecule, thus rendering it unable to bind hsp90. On thebasis of this hypothesis and to investigate the role of TPRdomains, we constructed the wild-type HBI I+II+11IBamH1 mutant (Fig. 3). Fig. 5 shows that the immunoreac-tive protein (lane 8) migrated with a slower mobility afterincubation with hsp90 (lane 9), whereas no modificationoccurred upon exposure to transferrin (lane 7), thus clearlydemonstrating formation of specific complexes. This latterfinding provides strong evidence that this new constructincluding the three intact TPR domains is able to associatewith hsp90 contrary to the mutated truncated construct.FK506 and Rapamycin Do Not Inhibit FKBP59-HBI Binding

to hsp9O. As reported recently, immunosuppressants neithermodify the composition of the heterooligomeric form ofprogesterone (14, 32) or glucocorticosteroid (33) receptorsnor affect reconstitution of the glucocorticosteroid receptorcomplex (34). Therefore we examined whether exposure toimmunosuppressive drugs would affect binding of FKBP59-HBI to hsp90. Although the binding ofrecombinant FKBP59-HBI to FK506-Afligel has been reported (14), we performedfluorescence studies to check if FKBP59-HBI could bind theimmunosuppressant. As already observed for FKBP12 (35),

1 2 3 4 5 6 7 8 9HSP9O - + - + - + - - +

FIG. 5. Mapping of the FKBP59-HBI hsp90-binding domain.HBI I+II (20 pmol; lanes 1 and 2), HBI I+II+IV (12.5 pmol; lanes3 and 4), HBI I+II+III M3 (24 pmol; lanes 5 and 6), or HBI I+II+IIIBamHl (25 pmol; lanes 8 and 9) without (-) or with (+) a 4-fold molarexcess of hsp90 was subjected to nondenaturing PAGE followed byimmunoblotting with EC1. Lane 7 corresponds to HBI I+11+IIIBamHl incubated with transferrin (100 pmol).

quenching of fluorescence occurred upon binding of FK506or rapamycin to FKBP59-HBI. After exposure of FKBP59-HBI to either FK506 or rapamycin prior to incubation withhsp90, the appearance of a complex migrating with a slowerelectrophoretic mobility led us to conclude that none of theimmunosuppressants affected binding ofthe immunophilin tohsp90 (data not shown). In addition, immunosuppressants didnot induce dissociation of preformed FKBP59-HBI-hsp90complexes (data not shown), but in this latter case, due to thepresence of tryptophan residues in the hsp90 sequence, nofluorescence studies have been carried out. Therefore wecould not provide any evidence for the binding of the drugsto preformed complexes.

DISCUSSIONTo obtain more information about the assumed associationbetween FKBP59-HBI and hsp90, we expressed FKBP59-HBI in the rabbit reticulocyte lysate, a suitable system thatmay facilitate identification of the domain(s) involved in thisinteraction. In the present work, our density gradient andgel-filtration data suggested that in vitro-translated full-lengthFKBP59-HBI is not associated with hsp90. Given that thepresence of rabbit FKBP59-HBI in the heat shock proteinheterocomplex including mouse hsp90 and hsp70 was re-ported to be stabilized if a steroid receptor was present (31),we also cotranslated estradiol receptor and FKBP59-HBI.Again and unlike the receptor, which was found as an 8-9Sentity, FKBP59-HBI migrated as a monomeric 3.5-4S form.Reconstitution of heat shock proteins binding to receptorswas also observed in an ATP-dependent manner (27-30).Although this observation remains intriguing because first,efficient in vitro dissociation of these proteins also requiresATP and divalent cations (27, 28), and second, the rabbitlysate already contains an ATP-generating system, in vitrotranslation was carried out in the presence ofthis system, butthe behavior ofFKBP59-HBI remained unaffected. Recentlyit has been shown that all ofthe FKBP59-HBI in reticulocytelysate is present in a large complex (31). The data reported inthis work are inconsistent with this observation since endog-enous rabbit FKBP59-HBI was not found associated withhsp90. Our inability to demonstrate specific interaction ofFKBP59-HBI with hsp90 in the reticulocyte lysate could beexplained by the fact that FKBP59-HBI may be in a weakcomplex with hsp90, which does not survive traditionalgradient centrifugation or gel filtration. Indeed, several au-thors have noted that FKBP59-HBI is clearly a more looselyassociated component of the receptor heterocomplex thanhsp90 (31, 34). In addition, many of the studies on thestructure of reconstituted steroid receptors have relied onimmunostaining to identify receptor components after immu-noadsorption under gentle conditions without carefully pu-rifying complexes. We used a similar approach to detectFKBP59-HBI-hsp90 complexes, but under these experimen-tal procedures, unlike hsp90, in vitro-translated FKBP59-HBI became bound to an irrelevant immunoadsorbent asalready reported for hsp70 (4, 36).

In the present work, the interaction of highly purifiedfull-length and truncated constructs of rabbit FKBP59-HBIand human hsp90, was assessed by nondenaturing gel elec-trophoresis. Upon incubation of wild-type full-lengthFKBP59-HBI with hsp90, we observed the appearance of aretarded band, suggesting formation of complexes. Immuno-staining with antibodies raised against both proteins clearlydemonstrated that this band reflected a specific and stablebinding of FKBP59-HBI to hsp90. Similar experiments car-ried out with mutants lacking the third domain of FKBP59-HBI (HBI I+II and HBI I+II+IV) indicated that this domainmay be involved directly or indirectly in hsp90 binding.Recent reports identified a TPR sequence in the 40-kDa

Proc. Natl. Acad. Sci. USA 91 (1994)

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kl. 40 0

Proc. Natl. Acad. Sci. USA 91 (1994) 11201

cyclophilin component of the unactivated estrogen receptor,which shares identity with FKBP59-HBI (11, 12). This motifcontains a characteristic 34-amino-acid repeat defined by adegenerate consensus sequence that could be divided intotwo subregions (corresponding to domains A and B) eachable to form short amphipathic a-helices (18, 19). Thisrepeated unit has been found in several proteins required forRNA synthesis, protein import, and Drosophila development(refs. 37 and 38 and references therein). The function oftheserepeats is unknown, but it has been postulated that they coulddirect protein-protein interactions (37, 38). Such interactionscould occur via the TPR sequence within the same proteinmolecule as well as between similar members of the TPRfamily or unrelated proteins. It is of interest to note that a60-kDa protein that interacts with hsp90 and hsp70 is also amember of the TPR family (39, 40). We have observed thatour full-length mutated construct failed to interact withhsp90. The fact that insertion of 2 amino acid residues in thesecond helix of the third TPR domain could modify thethree-dimensional structure of FKBP59-HBI, thus impedingits association with hsp90, prompted us to construct a newmutant containing the entire TPR sequence. Upon incubationwith hsp90, this mutant displayed a modified electrophoreticmobility, thus demonstrating the importance of the TPRmotifs in hsp90 binding. Whether these motifs interact di-rectly with hsp90 or modulate binding of another domainremains to be elucidated.We also investigated the effects of FK506 and rapamycin

on hsp90 binding. Our results suggest that exposure ofFKBP59-HBI to immunosuppressants does not impede as-sociation between the two proteins.

Altogether these findings indicate that both purified re-combinant proteins are able to fold into a conformationallowing formation of stable complex in an ATP-independentmanner as reported in another system (31).

In conclusion, our cell-free system should make studies ofmolecular interactions between FKBP59-HBI, hsp90, andsteroid hormone receptors possible. As cyclophilin 40 and aprotein of 60 kDa have also been found in untransformedsteroid receptor heterooligomers and contain TPR se-quences, it will be of interest to examine whether theseproteins also interact with hsp90. The fact that the TPR motifhas been found in proteins from different species and tissuessuggests that this specific sequence is of fundamental impor-tance. By binding to the TPR region of FKBP59-HBI, hsp90could prevent nonproductive interactions between the im-munophilin and other intracellular proteins. hsp90 may alsobe a substrate for the peptidyl-prolyl isomerase function ofsome immunophilins and in turn modify the function of otherproteins with which it interacts. The biological importance ofthe FKBP59-HBI-hsp90 interaction in the steroid receptorfunction remains to be elucidated.

We are grateful to Dr. N. Rebbe for the gift of the plasmid pKNI-3.We thank Dr. K. Murato (Fujisawa Pharmaceutical) for FK506 andDr. S. Sehgal (Wyeth) for rapamycin. We also thank Dr. L. E. Faberfor the EC1 monoclonal antibody, Dr. M. Sabbah and Dr. G.Redeuilh for helpful discussion throughout the course of this work,and Dr. K. Rajkowski for critical reading of the manuscript. Wegratefully acknowledge the help of J. C. Lambert and M. Bahloul fordrawing and photographs.

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