From the Structurally related tetratricopeptide repeat
motifs in steroid receptor-associated immunophilins and the STI1
homolog, Hop, mediate the interaction with a common cellular target,
hsp90. We have identified the binding domain in hsp90 for cyclophilin 40 (CyP40) using a two-hybrid system screen of a mouse cDNA
library. All isolated clones encoded the intact carboxyl terminus of
hsp90 and overlapped with a common region corresponding to amino acids 558-724 of murine hsp84. The interaction was confirmed in
vitro with bacterially expressed CyP40 and deletion mutants of
hsp90 Peptidyl-prolyl isomerases are cellular proteins that can mediate
changes in protein conformation by catalyzing cis-trans isomerization about amino acid-proline peptide bonds (1-3).
Immunophilins represent the predominant group within the rapidly
growing peptidyl-prolyl isomerase protein family that includes the
cyclophilins and FK506-binding proteins
(FKBPs)1 identified as
cellular targets for the immunosuppressant drugs cyclosporin A and
FK506, respectively (4, 5). An overlap of ligand binding and catalytic
domains in immunophilins results in an inhibition of isomerase activity
in response to immunosuppressant drug interaction (4, 5).
Cyclophilin 40 (CyP40) was first isolated and identified in association
with the unactivated estrogen receptor (6) and shares structural and
sequence homology with FKBP52, previously described as a common
component of aposteroid receptor complexes (7, 8). A third mammalian
immunophilin, FKBP51, with significant identity to CyP40 and FKBP52,
has recently been identified in the progesterone receptor complex (9).
The structural similarity among CyP40, FKBP51, and FKBP52 is
characterized by an amino-terminal immunophilin-like domain and a
conserved carboxyl-terminal tetratricopeptide repeat (TPR) domain that
mediates protein interaction (6, 9-12). Within steroid receptor
complexes the immunophilins bind competitively to heat shock protein
hsp90 to form distinct immunophilin-hsp90-receptor complexes (9, 13,
14). Conserved structural features that determine immunophilin
interaction with an identical site in hsp90 include the TPR domain
together with adjacent subregions located at the amino- and
carboxyl-terminal ends of the TPR domain, respectively (14, 15).
Despite the overall similarities between these immunophilins, there is
accumulating evidence that they possess unique structural elements
allowing distinct interactions with hsp90, leading to differential
responses of receptor function (9, 15, 16).
The dynamic assembly of steroid receptors to a high affinity hormone
binding conformation requires the cooperative interaction of the major
chaperones hsp70 and hsp90 (17-24; for review, see Ref. 25). Newly
synthesized receptor undergoes sequential refolding through reactions
mediated by hsp70, in concert with regulatory influences from the
co-chaperones hsp40, Hip, and Hop, leading to the formation of
receptor-hsp70-hsp90 complexes (23, 26-29). The conformation state and
chaperone function of hsp70 and hsp90 are controlled by nucleotide
binding (29-38). Hop provides the essential link between hsp70 and
hsp90 and binds the chaperones simultaneously through favored
interactions with ADP-bound forms of both proteins (37). In what is
likely to be a highly dynamic setting, Hop enhances the efficient
transfer of receptor substrate from hsp70 to hsp90 (28). After
dissociation of Hop and hsp70, ATP-dependent conversion of
hsp90 to the ATP-bound conformer promotes recruitment of p23 and one of
the immunophilins to generate mature receptor complexes (23-25, 28).
Although a precise role for p23 and the immunophilins in receptor
complexes has yet to be defined, evidence that these accessory proteins
are capable of independent chaperone activity (39, 40) raises the
possibility that they might modulate receptor function either directly
or indirectly via hsp90.
The majority of accessory proteins that participate in the
chaperone-mediated assembly of steroid receptors possess structurally related TPR motifs that mediate their interaction with hsp90 and/or hsp70 (11-14, 22, 41, 42). Thus, in addition to the large immunophilins, TPR units have been identified in the hsp70-binding protein Hip (29), and six repeats are present in the chaperone cofactor
Hop (41, 43). Distinct TPR domains located in the NH2- and
COOH-terminal regions direct the simultaneous interaction of Hop with
hsp70 and hsp90, respectively (22, 41). The ability of Hop to compete
effectively with the immunophilins for hsp90 binding (44), together
with similar observations with PP5, a serine protein phosphatase
recently identified as an additional component of the unactivated
glucocorticoid receptor (42), have led to suggestions of a common TPR
interaction site within hsp90 (44).
Genetic studies in yeast have confirmed that hsp90 is an essential
component for the activity of diverse intracellular signaling molecules
including steroid receptors and regulatory tyrosine kinases involved in
cell cycle control (for review, see Ref. 25). A role for CyP40 in hsp90
regulatory function has been demonstrated by studies in which deletion
of Cpr7, a Saccharomyces cerevisiae homolog of CyP40,
resulted in greatly reduced activities of both glucocorticoid and
estrogen receptors and pp60v-src kinase
expressed in yeast (45).
To date one of our approaches to determining the function of CyP40 and
its partner immunophilins has been to study their in vitro
interaction with the known chaperone components of steroid receptor
complexes. In this way we have identified hsp90 (14) and
hsp702 as major cellular
targets for CyP40 interaction. As an extension of this approach, we
have begun to use the yeast two-hybrid system (46) to search for
additional protein targets for CyP40. The modular domain structure of
the large immunophilins (6, 12, 47) makes it attractive to use these
domains separately as probes for protein interaction. In this way the
immunophilin domain of FKBP52 has been used successfully in a yeast
two-hybrid screen to isolate FAP48, a novel protein that may represent
a common natural ligand for FKBPs (48).
Here, we show that use of the yeast-based genetic assay, in combination
with the COOH-terminal TPR domain of CyP40 (amino acids 185-370), led
predominantly to the isolation of interacting clones that corresponded
to hsp90 cDNAs encoding the COOH-terminal dimerization domain of
hsp90. In vitro binding assays with deletion mutants of this
region located the interaction site for CyP40 to a 124-residue
COOH-terminal segment of hsp90 and confirmed that this region
incorporates the common TPR binding site for the immunophilins and Hop.
Deletion of the acidic EEVD motif, conserved at the carboxyl terminus
of hsp90 proteins, precluded CyP40 and Hop interaction, consistent with
a regulatory role of this microdomain in hsp90 function.
Plasmids--
pBTM116-CyP40 185-370 contains the carboxyl half
of CyP40 fused in-frame to the DNA binding domain of LexA (amino acids
1-202). The COOH-terminal portion of human CyP40, encoding residues
185-370, was amplified by PCR using primers that introduced an
EcoRI site at the 5'-end and a termination codon with an
EcoRI site at the 3'-end. Subcloning of this fragment into
pGEM 3Z (Promega) at the EcoRI restriction enzyme site
allowed confirmation of sequence integrity by automated sequence
analysis (Applied Biosystems). The EcoRI fragment was then
ligated into the corresponding restriction site in the bait vector
pBTM116 (49), in-frame with the LexA DNA binding domain to give
pBTM116-CyP40 185-370.
The plasmid for the protein GST-bCyP40 WT has been described (14). For
untagged wild-type bovine CyP40, a bCyP40 cDNA template was
amplified by PCR using sequence-specific oligonucleotide 5'- and
3'-primers containing NdeI and BamHI restriction
enzyme sites, respectively. A TGA stop codon was placed immediately
before the BamHI site. The PCR fragment was ligated into
pGEM-T (Promega), and the sequence integrity of both ends of the insert
in an isolated clone was confirmed by automated sequence analysis
(Applied Biosystems). An XhoI to BclI fragment
excised from a wild-type bCyP40 cDNA was replaced to eliminate
PCR-generated errors within this region. A full-length bCyP40 fragment
produced by NdeI and BamHI digestion was then
cloned into the pET-11 plasmid vector. The pET-11-bCyP40 WT expression
plasmid was transformed into the E. coli expression host
BL21(DE3). Overexpression of wild-type bovine CyP40 was induced by 0.4 mM IPTG, and lysates of recombinant CyP40 were prepared as
described below. Expression plasmids for human hsp90
The expression plasmid pET-28a(+) 589-732 Two-hybrid Screening--
Two-hybrid system reagents including
the mouse embryo cDNA library in pVP16, the S. cerevisiae reporter strain L40, and screening methodology were as
described previously (49, 52). Briefly, S. cerevisiae L40
cells were transformed sequentially with pBTM116-CyP40 185-370 and the
library, and the yeast transformants were plated on histidine-deficient
medium. Of the 40 × 106 yeast transformants screened,
some 300 were identified as histidine prototrophs of which 150 were
selected for assessment of Protein Expression and Purification--
The expression plasmid
for GST-bCyP40 WT fusion protein was transformed into E. coli strain XL-1 Blue, and the protein was purified from lysates
of bacterial cultures by affinity chromatography on glutathione-agarose
(Amersham Pharmacia Biotech) as detailed previously (14). The
expression plasmid for FKBP52 was transformed in E. coli
pVX28 (10). All other expression constructs used in this study were
transformed into E. coli BL21(DE3). Recombinant CyP40, Hop,
FKBP52, and hsp90
For expression of His-tagged hsp90 Protein Binding Studies--
Direct binding studies between
wild-type hsp90
In a reciprocal binding study, glutathione-agarose containing absorbed
GST-bCyP40 WT fusion protein was diluted 5-fold with Sepharose 4B, and
40-µl aliquots of the diluted gel were rotated separately for 3 h at 4 °C with 30 µg of the purified hsp90 truncated mutants
equilibrated in binding buffer (500 µl) containing 0.2% Triton
X-100. After pelleting by microcentrifugation, the gels were washed
repeatedly as already described, boiled in 2 × SDS-PAGE sample
buffer (40 µl), and then examined for protein retention by
SDS-PAGE.
To determine the binding preference of CyP40 for hsp90
The ability of Hop and FKBP52 to compete with CyP40 for binding to the
truncated mutant hsp90 581-724 Isolation of Murine Hsp84 cDNAs Encoding a Distinct Interaction
Domain for CyP40--
We used a modification (49, 52) of the yeast
two-hybrid system (46) to isolate cDNA clones encoding proteins
that bind to the COOH-terminal half of CyP40. This segment of CyP40
incorporates the TPR domain flanked by acidic and basic subdomains at
the NH2- and COOH-terminal ends, respectively (14). The
CyP40 COOH-terminal domain was fused to the LexA DNA binding domain,
and the resulting bait hybrid was used to screen a mouse embryo library
of hybrid proteins between the nuclear localized VP16 acidic activation domain and random cDNA fragments. Size selection for short
cDNAs (~ 500 nucleotides) facilitated rapid sequence analysis and
isolation of distinct protein interaction domains within full-length
polypeptides (52). Coexpression of the bait and target hybrids into the
yeast host S. cerevisiae L40 that contains two integrated
reporter constructs, the yeast HIS3 gene and the bacterial
lacZ gene, allowed the isolation of cDNAs coding for
putative CyP40-interacting proteins. With this approach, approximately
40 × 106 yeast transformants were screened. More than
300 histidine-positive prototrophs were detected, of which 150 were
assayed qualitatively for the presence of The Interaction Domain for CyP40 Is Located within the 124-residue
Carboxyl-terminal Region of Hsp90, and Deletion from the Carboxyl
Terminus of Hsp90 Interferes with CyP40 Recognition--
The mouse
hsp84 sequence of those clones found to interact most frequently with
CyP40 differs from the equivalent region of human hsp90
Our results with the yeast two-hybrid system determined that the
interaction domain for CyP40 was confined to a 200-residue COOH-terminal region of hsp90 corresponding to the dimerization domain
of the protein (Fig. 1). To confirm the ability of this region to
mediate CyP40 binding in vitro, we first prepared the truncated deletion mutant hsp90 530-724
We then prepared sequential NH2-terminal and COOH-terminal
deletions of this original construct to delineate further the
interaction domain for CyP40 (Fig. 2). The design of these additional
mutants was guided by information from available reports (57-59)
describing the microdomain structure within the hsp90 COOH-terminal
region (Fig. 2). For example, the construct hsp90 581-724
The ability of these truncated proteins to bind CyP40 was examined in
pull-down assays in which chelate-agarose charged with equal amounts of
wild-type hsp90
The results of Fig. 3, A and B, also indicated
that the 581-724 CyP40, FKBP52, and Hop Interact with Hsp90 via a Common Domain
within the Hsp90 Carboxyl Terminus--
Evidence that Hop and FKBP52
compete directly with CyP40 for hsp90 binding has led to proposals that
these proteins might target a common or overlapping site within hsp90
(44). It was of interest therefore to test the ability of Hop and
FKBP52 to bind to the COOH-terminal region of hsp90 which we had
determined to be important for CyP40-hsp90 interaction. Fig.
5 shows that incubation of our panel of
hsp90 deletion mutants, immobilized on chelate-agarose, with Hop
bacterial lysates produced a retention profile that closely resembled
the pattern observed with CyP40 (Fig. 3A). The result is
consistent with the presence of common binding elements for CyP40 and
Hop within a discrete carboxyl-terminal domain of hsp90. We next
examined the ability of FKBP52 and Hop to compete with CyP40 for
binding to hsp90 581-724 A search for cellular protein targets for the CyP40 COOH-terminal
protein-interaction domain (amino acids 185-370), using the yeast
two-hybrid method, resulted in the cloning of cDNAs encoding hsp90
with an intact carboxyl terminus. The overlapping region common to
these cDNAs corresponded to amino acids 558-724 of human hsp90 Binding analyses with sequential NH2-terminal deletion
mutants of the hsp90 530-724 After a comprehensive assessment of the effect of mutations throughout
chicken hsp90 Our study has demonstrated conclusively that CyP40 does not have a
binding preference for either hsp90 We have examined the 124-residue COOH-terminal sequence that
incorporates the TPR acceptor site in hsp90 to identify structural elements with potential to mediate hsp90 interaction with TPR proteins.
Charge distribution within this domain is highly conserved between
human hsp90 Several points of evidence suggest that the interaction site for Hop
and the TPR immunophilins lies within the dimerization domain of hsp90.
Dimerization is an intrinsic property of hsp90, required for its
biological function in intact cells (69). Nemoto et al. (56,
70) have demonstrated that the ability of hsp90 to dimerize and to form
higher order oligomers resides in the COOH-terminal 200 amino acids.
Furthermore, the same workers have proposed that dimerization of
hsp90 Evidence suggests that within steroid receptor heterocomplexes the
stoichiometry is defined by single molecules of receptor, p23 and one
of the immunophilins together with dimeric hsp90 (61). Although we have
not determined whether our hsp90 Shaknovich et al. (72) provided initial evidence that the
COOH-terminal domain of hsp90 might have a biological function beyond
that of dimer formation. They described the conversion of the basic
helix-loop-helix transcription factor MyoD to a form with enhanced DNA
binding activity through a transient interaction with a recombinant
protein containing the COOH-terminal 194 residues of hsp90 (72). This
conformational activation of MyoD occurred without the requirement of
ATP (72) and was extended to other basic helix-loop-helix proteins
(73). A recent analysis of human hsp90 Data from mutational analyses of hsp90 binding to steroid receptors are
consistent with the COOH-terminal half of hsp90 being involved in
receptor interaction and in the maintenance of receptor biological
function (64). Certain regions within hsp90 appear to have a
differential effect on hsp90-steroid receptor interaction and on the
activity of individual receptor systems (59, 68, 76, 77), highlighting
the diversity among the receptors. Deletion of the acidic region A
(residues 221-290) precluded the interaction of chicken hsp90 Hsp90 functions as a dimer and is divisible into three functional
domains (Fig. 8). The
NH2-terminal domain harbors a common site for ATP binding
and interaction with geldanamycin and has a chaperone activity that is
modulated by both agents (35, 36, 55, 74, 75). An ATP-independent
chaperone function exists in the COOH-terminal domain, a region that
also appears to be critically important for hsp90 dimerization (56, 68,
70, 71) and binding of client proteins such as steroid receptors (64,
68, 76, 77). Our results show that this domain is also a target for the
TPR-containing immunophilins and Hop. A central charged domain may
provide additional elements that contribute to stabilizing the dimeric
form of hsp90 (36) as well as interactions with steroid receptors
(57-59) and hsp90 partner proteins (16). Identification of an ATP
binding site within hsp90 has led to proposals that in response to ATP
binding and hydrolysis, hsp90 may undergo changes in conformation in a
manner analogous to hsp70 (36). Indeed, evidence suggests that these
conformational changes are translated throughout the hsp90 protein
(34-38) and may involve the MEEVD peptide at the hsp90 COOH terminus
(16). Thus only the ATP-bound state of hsp90 interacts with p23 (34).
Nucleotide exchange to the ADP-bound form stabilizes a hydrophobic
surface in hsp90 (34, 35) which may facilitate interaction with protein substrates (34-38). Hop binds preferentially to ADP-bound hsp90 and
blocks ATP-dependent conversion to a form capable of
interaction with p23 (37).
Department of Endocrinology and Diabetes,
Department of Biochemistry,
University of Western Australia, Nedlands Western Australia 6907, Australia, and the ** the Institute for Gene Therapy and Molecular
Medicine, the Mount Sinai School of Medicine,
New York, New York 10030
ABSTRACT
Top
Abstract
Introduction
References
and was delineated further to a 124-residue COOH-terminal
segment of hsp90. Deletion of the conserved MEEVD sequence at the
extreme carboxyl terminus of hsp90 precludes interaction with CyP40,
signifying an important role for this motif in hsp90 function. We show
that CyP40 and Hop display similar interaction profiles with hsp90 truncation mutants and present evidence for the direct competition of
Hop and FK506-binding protein 52 with CyP40 for binding to the hsp90
COOH-terminal region. Our results are consistent with a common
tetratricopeptide repeat interaction site for Hop and steroid
receptor-associated immunophilins within a discrete COOH-terminal domain of hsp90. This region of hsp90 mediates ATP-independent chaperone activity, overlaps the hsp90 dimerization domain, and includes structural elements important for steroid receptor interaction.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
(50), FKBP52
(10), and Hop were kindly provided as gifts by C. T. Walsh, D. A.
Peattie, and D. F. Smith, respectively. An identical cloning strategy
was used to generate NH2-terminal His-tagged expression
plasmids for hsp90
deletion mutants incorporating codons 530-724,
581-724, 600-724, 530-700, and 530-719. Each construct was PCR
amplified from pET-15b-hsp90
(50) full-length cDNA template
using Taq DNA polymerase with primers that introduced an
NdeI site at the 5'-end and a termination codon with an
NdeI site at the 3'-end. The PCR fragment was ligated into
pGEM-T and was then excised with NdeI digestion from a
suitable clone confirmed for sequence fidelity. The gel-purified
fragments were cloned into the pET-28a(+) vector that had been
linearized with NdeI to give the expression plasmids
pET-28a(+) 530-724
, pET-28a(+) 581-724
, pET-28a(+) 600-724
,
pET-28a(+) 530-700
, and pET-28a(+) 530-719
.
was prepared for the
NH2-terminal His-tagged hsp90
deletion mutant
encompassing residues 589-732. The plasmid was derived by PCR from a
full-length human hsp90
cDNA template (51) (kindly provided K. Yokayama) using Pfu DNA polymerase (Stratagene) and specific
oligonucleotide primers with built-in NheI (5'-end) and
BamHI (3'-end) restriction sites. The BamHI site
was preceded by a termination codon. Subcloning of the blunt ended PCR
fragment into SmaI-digested pGEM 3Z vector allowed
confirmation of sequence integrity. The fragment was excised with
NheI and BamHI and ligated into vector DNA to
give pET-28a(+) 589-732
.
-galactosidase activity. 28 His+,LacZ+ colonies were grown in liquid medium
deficient in leucine and were used to generate crude plasmid DNA
preparations. These were transformed into competent Escherichia
coli HB101 cells allowing selection of colonies containing library
hybrid plasmids. Purified minipreparations of the resultant clones were
transformed into L40 yeast cells simultaneously with pBTM116-CyP40
185-370 bait plasmid, and the transformants were assayed for
-galactosidase activity. To reduce the risk of false positives,
parallel transformations were performed with individual library
plasmids alone or in combination with
pBTM116,3 pBTM116-ARL-E1, and
pBTM-LYN as negative controls. cDNA inserts in library pVP16
plasmids, isolated from clones considered to be true positives, were
sequenced using both a sense (5'-GAGTTTGAGCAGATGTTTA-3') and antisense
M13 universal primer (Applied Biosystems). 10 murine hsp84-related
cDNAs (53) were recovered from the true positive clones.
proteins were overexpressed in bacterial cultures
by induction with IPTG. Lysates were prepared by sonicating bacterial
cells in lysing buffer (CyP40, Hop: 10 mM Tris, pH
7.3, containing 100 mM KCl, 1 mM
dithiothreitol, 0.2% v/v Triton X-100, and 1 mg/ml lysozyme; FKBP52
and hsp90
and
: 50 mM disodium hydrogen
orthophosphate, pH 7.4, containing 300 mM NaCl, 10 mM imidazole, 1 mM dithiothreitol, 0.2% v/v
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 1 mg/ml lysozyme) and were cleared of particulate material by
ultracentrifugation (100,000 × g for 30 min at
4 °C).
and
deletion mutants,
bacterial cells harboring these clones were pelleted from overnight cultures (150 ml) by centrifugation. After resuspension in 1.5 liters
of fresh 2 YT medium containing 200 µg/ml kanamycin, the bacteria
were incubated with shaking for a further 1 h at 37 °C. Protein
expression was induced with 1 mM IPTG over a 4-h period. Centrifugation gave a bacterial pellet that was resuspended in 20 ml of
lysing buffer (as described for FKBP52, hsp90
). The freeze-thawed
bacteria were sonicated on ice, and the lysates were recovered by
ultracentrifugation as already described. All His-tagged recombinant
proteins were purified from crude lysates by chromatography on
Ni-NTA-agarose (Qiagen). Unbound proteins were removed from the
chelate-agarose gel (0.5 ml) by 10 successive washes with 50 mM disodium hydrogen orthophosphate, pH 7.4 buffer, containing 300 mM NaCl, 10 mM imidazole, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride. The first wash also contained 1% v/v
Triton X-100. Gel charged with immobilized His-tagged proteins was
stored at 4 °C for use in protein binding studies, or the absorbed
protein was recovered by elution with the same buffer (1 ml) containing
500 mM imidazole. Recovered proteins were assessed for
purity by SDS-PAGE with Coomassie Blue staining, and after equilibration by dialysis against binding buffer (10 mM
Tris pH 7.3, containing 100 mM KCl and 1 mM
dithiothreitol) they were stored in 200-µl aliquots at
70 °C
until required.
and truncated hsp90 mutants with recombinant CyP40
and Hop were conducted in parallel. Briefly, chelate-agarose gels
containing immobilized hsp90-related proteins were equalized for
protein content by diluting up to 3-fold with Sepharose 4B. The diluted
gels were prepared in duplicate 50-µl aliquots, and each set of
duplicates was rotated for 3 h at 4 °C with IPTG-induced
bacterial lysates for CyP40 (150 µl) or Hop (200 µl). Both lysates
had been equilibrated previously in binding buffer plus 35 mM imidazole. The inclusion of imidazole to 35 mM concentration was found to be effective in limiting
nonspecific protein interaction with control chelate-agarose gel devoid
of hsp90-related proteins. The gels were subjected to replicate washes (8 × 500 µl) with binding buffer plus 35 mM
imidazole, the first 5 washes being supplemented with 0.2% v/v Triton
X-100. Gel-retained proteins were recovered by boiling in 40 µl of
2 × SDS-PAGE sample buffer and then analyzed by SDS-PAGE on
12.5% w/v polyacrylamide gel. Proteins were visualized by Coomassie
Blue staining.
versus hsp90
, the diluted glutathione-agarose gel
containing GST-bCyP40 WT fusion protein was suspended in 500 µl of
binding buffer plus 0.2% v/v Triton X-100, together with 30 µg of
protein consisting of either purified hsp90 530-724
or hsp90
589-732
alone or prepared in ratios of 1:1, 5:1, and 1:5,
respectively. After rotation for 3 h at 4 °C the gels were
washed free of unbound protein, boiled in 2 × SDS-PAGE sample
buffer (40 µl), and assessed for retention of the hsp90
and
mutants by SDS-PAGE.
was determined as follows. 0-, 20-, 50-, 100-, 200-, and 400-µl aliquots of induced Hop bacterial lysate
and 0-, 10-, 20-, 50-, 100-, and 200-µl aliquots of an extract
containing purified FKBP52 (concentration 0.54 µg/µl), were
supplemented with 30 µg of purified hsp90 581-724
protein and the
mixtures were brought to 500-µl total volume with binding buffer
containing 0.2% v/v Triton X-100. After a 3-h preincubation period at
4 °C, the mixtures were added to separate 40-µl aliquots of
Sepharose 4B-diluted glutathione-agarose containing immobilized GST-bCyP40 WT fusion protein. After rotation at 4 °C for a further 3 h the gels were washed as described, boiled with 40 µl of
2 × SDS-PAGE sample buffer, and analyzed for hsp90 581-724
protein retention by SDS-PAGE with Coomassie Blue staining. Protein was quantitated by densitometric scanning using Image Quant (Molecular Dynamics) software.
RESULTS
-galactosidase activity.
Transactivation of both reporter constructs was observed in 28 transformants, from which the library plasmids were isolated and
subjected to false positive analysis. These clones all interacted more
strongly with the CyP40 bait fusion protein than with several LexA
DNA-binding fusion controls. Sequence analysis of their cDNA
inserts revealed the presence of 10 incomplete murine hsp84
COOH-terminal clones, which fell into three distinct groups. Seven
inserts encoding residues 517-724 belonged to the most predominant
group. The remaining cDNAs encoded residues 501-724 (two clones)
and 558-724 (one clone), respectively (Fig.
1). These results strongly suggested that
the COOH terminus of hsp90 mediates binding to CyP40.
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Fig. 1.
Murine (mhsp84) isolates identified by
two-hybrid genetic screen with the COOH-terminal domain of CyP40.
Hsp90 functional domains for ATP/geldanamycin binding
(black) (43, 44, 70) and dimerization (shaded)
(56) are shown schematically. Numbers refer to amino acid
positions. CyP40 interaction isolates, denoted by amino acids
(AA) 501-724, 517-724, and 558-724, are represented as
narrow bars (black).
(53, 54) at
only five residues. Functional domains defined within hsp90 include a
common binding site for ATP/ADP and the antitumor agent geldanamycin in
the NH2 terminus (35, 36, 55) and a COOH-terminal region
that mediates hsp90 dimerization (56) (Fig. 1). Structural elements
include highly charged
-helical regions A and B and region Z, which
resembles a heptad repeat domain characteristic of leucine zippers
(57-59) (Fig. 2). These structural
domains have been predicted to play a role in hsp90-protein interaction
(57-59).
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Fig. 2.
Structural domains of
hsp90 and hsp90 COOH-terminal deletion
mutants. Structural elements for hsp90 include regions A, B, and Z
(57-59) and the MEEVD sequence motif at the extreme COOH terminus
(54). COOH-terminal truncation mutants for hsp90
and
isoforms
are represented as narrow bars (black).
incorporating an
NH2-terminal His-tag (Fig. 2). Pull-down assays in which
this protein was immobilized on Ni-NTA-agarose and then exposed to
bacterial lysate containing wild-type bCyP40 showed avid retention of
the cyclophilin (Fig. 3A,
lane 2). Moreover, in mapping studies (not shown) conducted with a series of GST-fusion proteins incorporating wild-type bCyP40 and
several CyP40 deletion mutants, the hsp90 530-724
protein displayed
an interaction pattern identical to that described previously for
full-length hsp90
(14). This COOH-terminal segment of hsp90 therefore appears to contain all of the essential elements for CyP40-hsp90 interaction.
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Fig. 3.
Binding properties of wild-type
hsp90 and hsp90
and
COOH-terminal truncation
mutants to CyP40. Panel A, chelate-agarose gels (50 µl) charged with His-tagged hsp90-related proteins were mixed with
rotation for 3 h at 4 °C with IPTG-induced bacterial lysate
(150 µl) for full-length bCyP40 in binding buffer containing 35 mM imidazole. After centrifugation, the gels were washed
repeatedly with the same buffer, initially supplemented with 0.2% v/v
Triton X-100. Gel-retained proteins were recovered with SDS-PAGE sample
buffer and analyzed on a 12.5% w/v polyacrylamide gel followed by
Coomassie Blue staining. Protein molecular weight markers (Amersham
Pharmacia Biotech) are shown on the left side. Panel
B, glutathione-agarose gels (40 µl) charged with GST-bCyP40 WT
fusion protein were rotated separately for 3 h at 4 °C with
purified hsp90 truncated mutants (30 µg) in binding buffer (500 µl)
containing 0.2% v/v Triton X-100. After microcentrifugation and
replicate washing, gel-bound protein was recovered with SDS-PAGE sample
buffer and analyzed as described for panel A.
was
prepared to assess the role of the hydrophilic region B in the
hsp90-CyP40 interaction. The EEVD motif, which is conserved at the
extreme COOH terminus in both the hsp70 and hsp90 molecular chaperone families (54, 60), has been shown to have an important regulatory role
in hsp70 function (60). Preparation of the hsp90 530-719
construct
was aimed at testing the influence of this regulatory motif on hsp90
recognition of CyP40. Although hsp90
and
appear to be equivalent
in the context of steroid receptor and immunophilin interaction (61),
we also prepared hsp90 589-732
to allow a direct comparison with
hsp90 581-724
for CyP40 binding efficiency.
and the recombinant deletion mutants was incubated
with bacterial lysates containing wild-type CyP40. Our binding profiles
showed that the hsp90 530-724
, 581-724
, and 600-724
proteins (Fig. 3A, lanes 2, 3, and
4) bound CyP40 with high efficiency. Removal of the
24-residue segment at the extreme COOH terminus of hsp90 (construct
530-700
) completely abolished CyP40 binding (Fig. 3A,
lane 6). Remarkably, the mutant 530-719
, in which the
last five residues MEEVD of hsp90
are deleted, was also unable to
interact with CyP40 (Fig. 3A, lane 5). The above
results were corroborated by a reciprocal study in which the hsp90
deletion mutants were incubated separately with GST-bCyP40 WT
fusion protein immobilized on glutathione-agarose (Fig. 3B).
Our results located the binding site for CyP40 to a 124-residue
COOH-terminal region of hsp90 and suggested that the presence of the
EEVD motif at the extreme COOH terminus of hsp90 is critical for
CyP40-hsp90 interaction.
and 589-732
deletion mutants were capable of
efficient interaction with wild-type CyP40. A more rigorous examination
of the possible preference of CyP40 for one or other of the hsp90
or
isoforms was conducted in a separate study. Analysis was
facilitated by the use of chelate-agarose-purified hsp90 530-724
and hsp90 589-732
deletion mutants, which allowed SDS-PAGE
discrimination on the basis of size. Fig.
4 shows the results of a binding study in which GST-bCyP40 WT, immobilized on glutathione-agarose, was exposed to
extracts containing either recombinant hsp90 530-724
(lane 1) or hsp90 589-732
(lane 2) alone or to mixtures
in which the ratios of the respective isoforms were 1:1 (lane
3), 5:1 (lane 4), and 1:5 (lane 5). It was
apparent from the observed binding profiles that CyP40 does not display
an affinity for one hsp90 isoform over the other.
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Fig. 4.
CyP40 does not show a binding preference for
hsp90 or
isoforms. Glutathione-agarose gels charged with GST-bCyP40
WT fusion protein were incubated as described in the Fig. 3B
legend with 30 µg of purified hsp90 530-724
or hsp90 589-732
alone or prepared in ratios of 1:1, 5:1, and 1:5, respectively. After
replicate washing to remove unbound protein, the gels were boiled in
SDS-PAGE sample buffer, and recovered proteins were analyzed by
SDS-PAGE.
. Extracts containing the purified hsp90
deletion construct were preincubated with increasing amounts of
purified FKBP52 or Hop bacterial lysate. After addition of
glutathione-agarose charged with GST-bCyP40 WT fusion protein, the
mixtures were incubated for a further period, and the gels were then
assessed for retention of hsp90 581-724
by SDS-PAGE. Fig.
6 shows that increasing concentrations of
FKBP52 caused a progressive reduction in the relative amount of the
hsp90 deletion mutant retained by gel-immobilized CyP40. Similar
competitive binding was observed with Hop (not shown). Together the
results confirmed that CyP40, FKBP52, and Hop interact directly with a common domain in hsp90 581-724
.
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Fig. 5.
Interaction properties of wild-type
hsp90 and hsp90
and
COOH-terminal truncation mutants
with Hop. Chelate-agarose gels (50 µl) charged with His-tagged
hsp90-related proteins were rotated for 3 h at 4 °C with
IPTG-induced bacterial lysate (200 µl) for Hop in binding buffer
containing 35 mM imidazole. After microcentrifugation and
replicate washing, the gels were assessed for bound Hop by SDS-PAGE as
described in the Fig. 3A legend.
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Fig. 6.
FKBP52 competition with CyP40 for binding to
hsp90 581-724 . Aliquots (0, 10, 20, 50, 100, and 200 µl) of purified FKBP52 extract (FKBP52 concentration
0.54 µg/µl) were supplemented with purified hsp90 581-724
protein (30 µg) and were equalized to 500 µl with binding buffer
plus 0.2% v/v Triton X-100. After brief mixing, the samples were
preincubated for 3 h at 4 °C and then added to separate 40-µl
aliquots of glutathione-agarose charged with GST-bCyP40 WT fusion
protein. After rotation at 4 °C for a further 3 h, the gels
were washed as in Fig. 3B and analyzed for retained hsp90
581-724
by SDS-PAGE as already described.
DISCUSSION
.
To confirm this interaction in vitro, the deletion mutant
hsp90 530-724
, containing an NH2-terminal histidine
tag, was immobilized on chelate-agarose gel and shown to retain
wild-type bCyP40 specifically. Pull-down assays with recombinant hsp90
530-724
and GST-CyP40 deletion mutants on glutathione-agarose
revealed a binding profile similar to that determined previously for
full-length hsp90 (14), suggesting that the COOH-terminal hsp90 segment contains the essential binding elements for CyP40-hsp90 interaction.
protein showed the recognition site
for CyP40 to be located within a 124-residue, COOH-terminal region of
hsp90. A deletion mutant truncated from the COOH-terminal end by
deleting the pentapeptide MEEVD was unable to bind CyP40, highlighting the critical importance of this conserved motif for CyP40-hsp90 interaction. Under the same conditions, the interaction profile of Hop
for the hsp90 derivatives closely matched that displayed by CyP40.
This, together with evidence of direct competition of Hop and FKBP52
with CyP40 for binding to hsp90 581-724
protein, supports the
existence of a common TPR interaction site for Hop and the
immunophilins within a discrete COOH-terminal domain of hsp90. It is
possible, however, that additional regions in hsp90, lying outside of
this general TPR acceptor site, contribute to stabilize the interaction
between hsp90 and its cochaperone partners (16). Our data extend the
findings of recent reports by Chen et al. (16) and by Young
et al. (63) that Hop and the steroid receptor-associated
immunophilins bind to the COOH-terminal region of hsp90.
on hsp90 interaction with accessory proteins, Chen
et al. (16) have been able to provide detailed comparisons
between the hsp90 binding requirements for Hop, the TPR-containing
immunophilins, and p23. Replacement of the EEVD motif by AAVD, at the
extreme COOH terminus of hsp90, reduced hsp90 interactions for all of
the TPR proteins, with binding being all but eliminated for CyP40 and
Hop (16). The interactions of hsp90 with FKBP52 and FKBP51 were less
sensitive to this mutation, with significant binding levels being
maintained (16). Our study, in which the hsp90 mutant deleted in the
MEEVD peptide failed to recognize both CyP40 and Hop, is consistent
with the findings of Chen et al. (16). It is possible that,
as in hsp70 (60), the conserved EEVD motif might contribute to the
overall conformation of the hsp90 protein and have a role in the
intramolecular regulation of hsp90 function.
or
. From heat-induced dissociation experiments with FKBP52·hsp90 complexes, Czar et al. (61) noted previously that neither of the isoforms is
dissociated selectively, suggesting that FKBP52 may bind equivalently
with each hsp90 subtype. The isoforms are produced in equal amounts in
higher eukaryotes (64) and share an 86% homology, differing at 99 residues in their amino acid sequences (54). A glutamate-rich segment
within the NH2-terminal sequence distinguishes the hsp90
class from hsp90
homologs (54). Heat shock induces a more profound increase in hsp90
expression compared with hsp90
, and only
hsp90
is induced by adenovirus E1A (65). Despite these differences, virtually identical hydropathy plots for the isoforms indicate a high
degree of structural similarity (54). It is of interest, however, that
the hsp90 species associated with chicken steroid receptors (66) and
that isolated with the bovine estrogen receptor (67) have both been
identified as
isoforms.
and
(54) and in chicken hsp90
(66) and mouse
hsp84 (53). Taking this charge distribution into account, we divided
the binding region in hsp90
into two subdomains (Fig. 7). A highly acidic region is located at
the carboxyl terminus (amino acids 691-724, 1 basic and 15 acidic
residues) and includes the EEVD motif, deletion of which abrogates
hsp90-TPR protein binding. An essentially neutral subdomain (amino
acids 600-690, 16 basic and 12 acidic residues), which is
characterized by a repeated helix-coil-helix secondary structure (57),
contains a concentration of charged residues between amino acids
623-653 (9 basic and 7 acidic residues) (54). This hydrophilic domain sits adjacent to a hydrophobic region (amino acids 657-673 in human
hsp90
, equivalent to amino acids 661-677 in chicken hsp90
) which
is necessary for hsp90 dimerization (68). Chen et al. (16)
have also shown that deletion of this 16-residue segment causes a
marked reduction in the binding of hsp90 to Hop and the steroid
receptor-associated immunophilins. Removal of this critical domain
disrupts a predicted
-helical structure (57), possibly resulting in
conformational changes unfavorable for dimerization and TPR-protein
interaction.
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Fig. 7.
Structure of the TPR interaction domain in
hsp90. The TPR acceptor site of hsp90 is located within a
COOH-terminal region spanning amino acid residues 600-724. Acidic and
basic residues are highlighted by negative and positive
charges, respectively. Within this sequence exists an acidic domain,
defined by residues 690-724, which includes the EEVD sequence motif at
the extreme COOH terminus. A 50-residue segment (623-673) incorporates
a hydrophilic domain adjacent to a hydrophobic region (residues
653-673) and includes two predicted
-helical microdomains
(57).
is mediated by the interaction of one subdomain (defined by
residues 542-615) with a different subregion (residues 621-698) of a
second hsp90 subunit (56, 71). Such a mechanism is compatible with the
observed sensitivity of hsp90-TPR protein interaction to deletions
within these specific regions (16).
mutants exist as dimers or
monomers, the protein hsp90 600-724
is largely deficient in
sequences thought necessary for dimerization (56, 71) and is therefore
likely to be monomeric. Efficient binding of both CyP40 and Hop to this
mutant suggests that both proteins are capable of interacting with a
monomer of hsp90.
NH2-terminal
(residues 9-236) and COOH-terminal (residues 629-732) domains has
confirmed independent chaperone activities within these sites (74, 75).
In full-length hsp90, the sites appear to contribute independently to
chaperone activity, differing in substrate specificity and nucleotide
dependence (74, 75). The presence of chaperone and dimerization
functions within a discrete hsp90 COOH-terminal domain, which also
mediates interactions with TPR cochaperone proteins, is intriguing. It
is possible then that hsp90 partner proteins, such as Hop and the
TPR-containing immunophilins, might have an important modulating role
in hsp90 function (38).
with
the glucocorticoid receptor (59), but recognition of the progesterone
(76) and estrogen receptors (68) was unaffected. Critical regions for
hsp90-progesterone receptor binding are located between residues 381 and 441, and 601 and 677 of chicken hsp90
(76). The first
incorporates the leucine heptad repeat region Z (59), and the second
overlaps the hsp90 dimerization domain (56). Deletions that compromise the in vivo interaction of hsp90 with the estrogen receptor
include those that correspond to region Z and the charged region B
(residues 521-567). Additional elements implicated in hsp90
association with the estrogen receptor are contained between residues
601 and 620, and 698 and 728 (68). The latter defines the acidic subdomain at the carboxyl terminus of hsp90 (57, 66). Its deletion
results in the loss of hsp90 dimerization (68). Deletion of the
hydrophobic sequence between residues 661 and 677 also disrupts hsp90
dimer formation, but this segment appears to be dispensable for
hsp90-estrogen receptor interaction (68).
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Fig. 8.
Hsp90 functional domains. The
NH2-terminal sequence (residues 1-221) incorporates a
chaperone function (74, 75) and binding sites for geldanamycin and ATP
(35, 36, 55). A dimerization domain (residues 524-724) exists at the
COOH-terminal end of hsp90 (56). This overlaps a region (residues
621-724) that elicits a chaperone function (74, 75) and the ability to
interact with TPR proteins. A large segment (residues 221-724)
includes elements that mediate association with steroid receptors (64,
68, 76, 77).
It is possible that the interaction between hsp90 and different target
substrates is governed by different cochaperones (38). The three
immunophilins CyP40, FKBP51, and FKBP52 then might modulate the
function of hsp90 either by altering hsp90 conformation or by
influencing ATP binding and the ability of hsp90 to recognize and
interact with unfolded substrate proteins (34, 38). A precedent for
such controlling influences over hsp90 has been set by the ability of
CyP40 to inhibit c-Myb DNA binding activity via a mechanism that
requires both the CyP40 protein interaction domain and its
peptidyl-prolyl isomerase function (62). In an alternate model, hsp90
may provide a scaffold for TPR proteins, allowing them to locate close
to hsp90 chaperone substrates. From such a position the immunophilins
CyP40, FKBP51, and FKBP52 might act directly on steroid receptors to
modulate receptor activity through an independent chaperone function
(39, 40).
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ACKNOWLEDGEMENTS |
---|
We offer special thanks to Drs. C. T. Walsh,
D. A. Peattie, and D. F. Smith for expression plasmids for human
hsp90, FKBP52, and Hop, respectively, and to Dr. K. Yokoyama for a
plasmid containing human hsp90
cDNA.
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FOOTNOTES |
---|
* This work was supported by the National Health and Medical Research Council of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Verdun
Street, Nedlands W.A. 6009, Australia. Tel.: 61-8-9346-2596; Fax:
61-8-9346-3221; E-mail: tomr{at}cyllene.uwa.edu.au.
The abbreviations used are:
FKBPs, FK506-binding
proteins; CyP40, 40-kDa cyclophilin; bCyP40, bovine CyP40; TPR, tetratricopeptide repeat; PCR, polymerase chain reaction; GST, glutathione S-transferase; WT, wild-type; IPTG, isopropyl
-D-thiogalactopyranoside; PAGE, polyacrylamide gel electrophoresis.
2 A. Carrello, manuscript in preparation.
3 E. Ingley, manuscript in preparation.
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REFERENCES |
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