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J. Biol. Chem., Vol. 275, Issue 23, 17857-17862, June 9, 2000
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From the Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688
Received for publication, February 28, 2000, and in revised form, March 31, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The sequential binding of different
tetratricopeptide repeat (TPR) proteins to heat shock protein 90 (hsp90) is essential to its chaperone function in vivo. We
have previously shown that three basic residues in the TPR domain of
PP5 are required for binding to the acidic C-terminal domain of hsp90.
We have now tested which acidic residues in this C-terminal domain are
required for binding to three different TPR proteins as follows: PP5,
FKBP52, and Hop. Mutation of Glu-729, Glu-730, and Asp-732 at the C
terminus of hsp90 interfered with binding of all three TPR proteins.
Mutation of Glu-720, Asp-722, Asp-723, and Asp-724 inhibited binding of FKBP52 and PP5 but not of Hop. Mutation of Glu-651 and Asp-653 did not
affect binding of FKBP52 or PP5 but inhibited both Hop binding and
hsp90 chaperone activity. We also found that a conserved Lys residue
required for PP5 binding to hsp90 was critical for the binding of
FKBP52 but not for the binding of Hop to hsp90. These results suggest
distinct but overlapping binding sites on hsp90 for different TPR
proteins and indicate that the binding site for Hop, which is
associated with hsp90 in intermediate stages of protein folding,
overlaps with a site of chaperone activity.
Heat shock protein 90 (hsp90)1 is a molecular
chaperone required for the proper folding of steroid receptors and a
growing number of proteins involved in signal transduction (1, 2). Over
the past few years, many co-chaperones that play an important role in
the folding of different subsets of proteins via the hsp90 pathway have
been identified (1, 2). Several of these proteins bind to hsp90 via
tetratricopeptide repeat (TPR) domains. The order and specificity with
which different TPR proteins bind to hsp90 complexes containing newly
synthesized proteins is critical for proper protein folding (3, 4).
This pathway has been best characterized in the study of steroid
receptors. Newly synthesized receptors are found in complexes with
hsp70. Intermediate complexes contain hsp70, hsp90, and Hop, a protein
with two separate TPR domains that bind to hsp70 and hsp90. At this
stage, receptors acquire the ability to bind hormone. Mature complexes
contain either PP5 or one of the large immunophilins (FKBP51, FKBP52, or CyP-40), each of which contains a single hsp90-binding TPR domain
(reviewed in Refs. 1-4). At this point, the receptor can bind steroids
with high affinity and is fully functional in vivo.
Understanding the structural basis for the interactions between TPR
proteins and hsp90 has become an important problem. Not only must
specific TPR proteins bind to hsp90 complexes at specific stages of
protein folding, but the TPR proteins found in mature hsp90 complexes
containing steroid receptors differ according to the identity of the
receptor (2). In the case of the glucocorticoid receptor, the identity
of the large immunophilin bound can dramatically affect hormone binding
affinity (5). Furthermore, PP5 has been shown to modulate
glucocorticoid receptor signaling (6, 7). Thus, specificity for the
binding of TPR proteins to hsp90 exists and is functionally important,
but its structural basis remains unknown. Determining what guides a
particular TPR protein to a particular hsp90 complex at a particular
time is of interest not only in terms of understanding protein folding
but also for potential drug development. Geldanamycin is an antitumor
antibiotic that blocks hsp90 function by competing with ATP for a
binding site at its N terminus (8, 9). Its biological activity is
thought to be due to prevention of proper folding and stabilization of proteins required for cell growth. If it were possible to specifically inhibit the binding to hsp90 of Hop, of PP5, or of particular large
immunophilins, it would be possible to target specific subsets of
proteins involved in cell signaling.
Hsp90 contains two functional chaperone domains. One domain, at the N
terminus, has been extensively characterized, including determination
of its three-dimensional structure (8, 9). The structure of the other
domain, at the C terminus, is not known. This highly acidic 12-kDa
domain contains the binding site for TPR proteins and can promote
protein folding in an ATP-independent manner (10-12). Although the
structure of this domain has not yet been determined, the structure of
the TPR domain of PP5, to which it binds, has been solved (13). The TPR
domain of PP5 consists of a series of antiparallel We have shown that, for PP5, four basic residues in this groove are
involved in binding to the C-terminal domain of hsp90 (16). Several of
these basic residues are conserved in Hop and in the large
immunophilins. We have now used site-directed mutagenesis to determine
the location of both the TPR-binding and chaperone sites within the
C-terminal domain of hsp90. Our results indicate that similarities
exist between the binding sites for PP5, FKBP52, and Hop, but we also
identified interesting differences between these binding sites. Thus,
the structural basis for the different biological activities of these
different TPR proteins may lie partly in different interactions between
their TPR domains and hsp90.
Plasmid Construction--
Sequences encoding rabbit FKBP52 and
murine Hop were amplified by polymerase chain reaction, using plasmids
pGEM-7Zf-p59 (17) and pGEX3X2000 (18) as templates. This reaction
introduced a 5' KpnI site, a 3' XbaI site, and
sequences encoding an N-terminal FLAG tag (DYKDDDDK) into both FKBP52
and Hop. Polymerase chain reaction products were cloned into the
corresponding sites of Bluescript SK(+) and, after sequencing to
confirm the absence of undesired mutations, subcloned into pCMV5 (19)
for expression in mammalian cells. Construction of pCMV6-FLAG-PP5 has
been described previously (6).
Site-directed Mutagenesis--
Mutagenesis with the Stratagene
QuikChange kit was performed according to the manufacturer's
instructions. For C90, the previously described pET30a-C90 (16) was
used as a template to mutate the indicated residues to Ala. The
Bluescript constructs described above were used as templates to mutate
Lys-354 of FKBP52 and Lys-429 of Hop to Ala. All mutants were sequenced
to confirm the presence of the desired mutations and the absence of
additional mutations. The FLAG-tagged Hop and FKBP52 mutants were then
subcloned into pCMV5 as described above.
Tissue Culture and Transfections--
COS-7 cells were
maintained in Dulbecco's modified Eagle's medium containing 10%
fetal bovine serum and 5 µg/ml gentamicin. Transfections were
performed using Superfect reagent (Qiagen) according to the
manufacturer's instructions.
Co-immunoprecipitation of Hsp90 with TPR Proteins--
Two days
after transfection of COS-7 cells in 60-mm plates with the indicated
plasmids, cells were washed on ice with 20 mM Hepes, pH
7.4, 150 mM NaCl (HBS), and scraped into 1 ml of 10 mM Hepes, pH 7.4, 20 mM sodium molybdate, 10 mM MgCl2, 1 mM EDTA containing 10 µg/ml each aprotinin and leupeptin. Cells were lysed by passage 10 times through a 26-gauge needle. Lysates were clarified by
centrifugation at 4 °C for 20 min at 21,000 × g. To
reduce nonspecific binding, supernatants were incubated with
end-over-end rotation for 1.5 h at 4 °C with 25 µl of
anti-mouse IgG-agarose beads (Sigma). After removing the beads by
centrifugation, FLAG-tagged TPR proteins were adsorbed to 25 µl of M2
beads (Sigma) for 1.5 h at 4 °C with end-over-end rotation.
After five washes with HBS containing 1% Triton X-100 (HBST), beads
were heated in SDS sample buffer, and samples were analyzed by
immunoblotting with monoclonal antibodies to hsp90 or to the FLAG
epitope as described previously (16).
Purification of C90 Proteins--
800-ml cultures of BL21 (DE3)
cells containing the various pET30a-C90 constructs were induced with
0.25 mM
isopropyl-1-thio- Binding of TPR Proteins to Wild-type and Mutant C90--
Two
days after transfection with the indicated plasmids, COS-7 cells in
10-cm plates were placed on ice, washed with cold HBS, and lysed in 1 ml of HBST containing 10 µg/ml each aprotinin and leupeptin.
FLAG-tagged proteins from clarified lysates were adsorbed to M2 beads
as described above, followed by washing 4 times with HBST. Purified C90
proteins, diluted to 50 µg in 200 µl of HBST and preadsorbed with
25 µl of Sepharose 4B beads (Sigma), were then incubated with the
washed M2 beads for 1.5 h at 4 °C with end-over-end rotation.
After 5 washes with cold HBST, beads were heated in sample buffer, and
proteins were separated by SDS-PAGE and transferred to nitrocellulose
membranes. To detect the S-tagged C90 proteins, membranes were probed
with S-protein conjugated to HRP (Novagen) according to the
manufacturer's instructions. An aliquot of each sample was also
analyzed by immunoblotting with M2 to ensure equal recovery of
FLAG-tagged TPR proteins.
In similar assays the C90 proteins rather than the TPR proteins were
immobilized. The purified C90 proteins were diluted to a final
concentration of 0.25 mg/ml in HBST containing 10 mM
imidazole, and 50 µg of each sample was allowed to bind to 25 µl of
His·Bind beads (Novagen) followed by washes with the same buffer.
Lysates from transfected COS-7 cells were prepared as described above but with 35 mM imidazole added to the lysis buffer. The
lysates were incubated for 1.5 h at 4 °C with the immobilized
C90 proteins, with end-over-end rotation. After 5 washes with HBST
containing 10 mM imidazole, beads were heated in sample
buffer and samples analyzed by blotting with M2 or S-protein as
described above.
Chaperone Assays--
The ability of C90 proteins to inhibit
rhodanese aggregation was assayed essentially as described (11, 21).
C90 proteins were equilibrated for 5 min at 25 °C in 990 µl of 50 mM Hepes, 100 mM NaCl, pH 7.0, at
concentrations of 0, 2, 4, and 10 µM. Ten µl of 50 µM rhodanese in 6 M guanidine HCl, 50 mM Hepes, pH 7.0, was then added, and the rate of rhodanese
aggregation was determined by measuring the increase in absorbance at
320 nm. Aggregation was essentially complete after 20 min.
Similarities and Differences between TPR-binding Sites for
Hsp90--
We have previously shown that four basic residues (Lys-32,
Arg-74, Lys-97, and Arg-101) located in a groove in the TPR domain of
PP5 are important for binding to the acidic C terminus of hsp90 (16).
Since these residues are conserved in the TPR domains of FKBP52 and Hop
(Fig. 1), we hypothesized that they would
also be important for the binding of these proteins to hsp90. The
sequences of the TPR domains of PP5, FKBP52, and Hop, all of which bind to hsp90, are aligned in Fig. 1, with the residues important for PP5
binding to hsp90 highlighted. Lys-97 of PP5 is absolutely required for
binding of the TPR domain of PP5 to hsp90 (16), but the role of
homologous residues in the binding of other TPR proteins to hsp90 had
not previously been tested. We therefore mutated the corresponding
residue in FKBP52 and in Hop. A co-immunoprecipitation assay was then
used to test the ability of the wild-type and mutant TPR proteins to
bind to hsp90 in transfected COS cells.
As shown in Fig. 2, hsp90
co-immunoprecipitated as expected with FLAG-tagged PP5, FKBP52, or Hop
from lysates of transfected COS-7 cells. When Lys-97 in PP5, or the
corresponding Lys-354 in FKBP52 was mutated to Ala, the interaction
with hsp90 was abolished (Fig. 2). Mutation of the corresponding
Lys-429 in Hop, however, did not reduce binding to hsp90 (Fig. 2).
These results suggested both similarities and differences between the
hsp90-binding determinants for FKBP52, PP5, and Hop. Alternatively, it
is possible that the sequence alignments are misleading and that
Lys-429 in Hop may prove not to correspond to Lys-97 of PP5 when the
structure of the Hop TPR domain has been determined.
If different residues in different TPR proteins are involved in binding
to hsp90, however, it stands to reason that different residues in hsp90
may be involved in binding to different TPR proteins. We therefore
compared which hsp90 residues were involved in binding to PP5,
FKBP52, and Hop.
Identification of Acidic Residues in Hsp90 Required for Binding of
TPR Proteins--
We and others (10, 16, 22) have shown that the
C-terminal 12-kDa fragment of hsp90, designated C90, contains the
binding site for the TPR domains of PP5, FKBP52, CyP-40, and Hop.
Acidic residues, in particular the EEVD sequence at the C terminus of hsp90, have been implicated in these interactions (16, 22, 23). In
order to identify which of the 25 acidic residues in C90 are required
for interaction with TPR proteins, we mutated each of these residues to
Ala, either singly or in groups, in a series of 13 mutations (Fig.
3). The ability of each mutant C90
protein to bind to PP5, FKBP52, and Hop was then examined.
FLAG-tagged TPR proteins were overexpressed in COS-7 cells and purified
from lysates by adsorption to anti-FLAG beads. The beads, with bound
TPR proteins, were then incubated with purified wild-type C90 or C90
mutants. After washing the beads to remove unbound material, samples
were analyzed by SDS-PAGE and blotting with S-protein, to detect the
S-tagged C90 proteins. As a control, parallel blots were developed
using the M2 anti-FLAG antibody to determine the level of the bound TPR
proteins. Mutations 8 and 9 were not characterized because they
aggregated and bound nonspecifically to beads (data not shown).
As shown in Fig. 4 (top), the
only mutation that significantly affected the binding of C90 to
immobilized full-length PP5 was mutant 13, in which the C-terminal EEVD
sequence was mutated to AAVA. This mutation completely blocked binding.
Each of the binding assays presented here was performed at least twice,
and other small quantitative differences in binding were not
reproducible. As expected based on sequence homology to the TPR domain
of PP5, and on a known interaction with the MEEVD sequence of hsp90
(23), results were similar with FKBP52 (Fig. 4, middle). In
this case, both mutation 12 and mutation 13 inhibited C90 binding.
These mutations, however, resulted in only partial inhibition of
binding to FKBP52, unlike the complete elimination of binding to PP5
observed with mutant 13. In both cases, however, the critical binding
determinants were at the very C terminus of hsp90. This was consistent
with previous studies suggesting a role for the C-terminal MEEVD
sequence in binding several different TPR proteins, and with this
sequence being somewhat less important for the binding of FKBP52
binding than for that of other proteins (23).
Quite different results were obtained when we examined the ability of
the panel of C90 mutants to bind to immobilized Hop (Fig. 4,
bottom). In this case, the only mutation that blocked binding was mutant 4, located toward the amino end of C90. The binding
of mutant 2 to Hop was also somewhat less than that of wild-type C90
(Fig. 4, bottom), and this decreased binding was seen in two
separate experiments (data not shown). Mutations at the C terminus had
no obvious effect on binding to Hop. Thus, different determinants on
hsp90 seemed to be involved in binding to different TPR proteins.
This last result was surprising in that, using different materials and
methods, two other groups have reported that the MEEVD sequence was
required for Hop binding (22, 23). It was difficult to compare our data
with those of Chen et al. (23) due to numerous differences
in experimental design. The only major difference between the methods
of Carello et al. (22) and ours, however, seemed to be that
their binding assays were performed with immobilized C90 proteins and
soluble Hop, whereas we had examined the binding of soluble C90 mutants
to immobilized Hop. We wondered whether this simple difference in assay
protocols might explain the divergent results.
We therefore used the methods described in Ref. 22 to examine the
ability of soluble FLAG-tagged PP5, FKBP52, and Hop, from COS cell
extracts, to bind to C90 proteins immobilized via their His tags (Fig.
5). By using this assay we obtained
similar results to those described above for PP5 and for FKBP52 (Fig.
5, top and middle), except that binding was even
more sensitive to the two C-terminal mutations. Mutant 13 did not bind
at all to either protein, and binding of mutant 12 was barely
detectable. Thus, using either binding assay, it is clear that critical
binding determinants for FKBP52 and for PP5 reside primarily at the C terminus of the C90 protein. The situation was more complex for Hop;
soluble Hop bound only weakly to immobilized mutant 4, consistent with
the results shown above. It did not bind at all, however, to
immobilized mutant 13 (Fig. 5, bottom). Thus, conformational changes or steric hindrances, resulting from protein immobilization in
the binding assay, are probably responsible for the observed differences in C90 residues required for Hop binding. We suggest that
Hop interacts with determinants altered in mutants 2, 4, and 13. We
further conclude that when Hop is immobilized, some binding
determinants become inaccessible, making the residues altered in
mutants 2 and 4 more important for binding. Similarly, when C90 is
immobilized, we speculate that other C90 residues become inaccessible
to Hop, making the residues altered in mutant 13 critical for binding.
Under ordinary circumstances, multiple sites of interaction may make
any single determinant dispensable.
For all three immobilized TPR proteins we also observed enhanced
binding of mutants 6 and 7 relative to the wild-type C90 (Fig. 4).
Unlike mutants 8 and 9, this did not appear to be due to nonspecific
binding to the beads (data not shown) but was not seen in assays using
immobilized C90 (Fig. 5). The significance of this observation is thus
unclear. The effects of all of the hsp90 mutations are summarized in
Table I.
Role of Acidic Residues in the Chaperone Activity of C90--
It
has been shown that hsp90 contains two independent chaperone sites, one
present at the amino end of the molecule, and one present in the C90
fragment (11, 12). We used our panel of C90 mutants to examine how the
site of chaperone activity compared with the sites of TPR binding. The
ability of C90 and of the C90 mutants to inhibit the aggregation of
rhodanese was determined essentially as described previously (11, 21).
Mutants 8 and 9 were not examined, due to aggregation and due to the
inability to express and purify sufficient amounts of mutant 9. As
shown in Fig. 6, and summarized in Table
I, mutations near the C terminus of C90 had little or no effect on
chaperone activity. Whereas mutants 3 and 7 appear to have increased
chaperone activity, only mutation 4 significantly interfered with the
ability of C90 to inhibit rhodanese aggregation. Interestingly, this is
the same mutation that inhibited the binding of C90 to Hop but not to
the other TPR proteins.
We have previously identified a groove in the TPR domain of PP5
that contains several basic residues critical for the binding of hsp90,
and we hypothesized that these basic residues interacted with the
abundant acidic residues in the 12-kDa C-terminal domain of hsp90 (16),
designated C90. It has been speculated that this groove could bind an
amphipathic At least two caveats apply to these data. First, as always, there is
the possibility that site-directed mutagenesis can have global effects
on protein folding, i.e. that particular mutants may result
in altered folding of either C90 or of the TPR proteins. Although
determination of the three-dimensional structure of hsp90 complexes
with these proteins will be required to address fully this question, it
is clear that whether due to loss of charged residues or whether to
altered folding TPR proteins do not bind to hsp90 in an identical
fashion. Similarly, homologous mutations in three TPR proteins
differentially affect their binding to hsp90. We are, therefore,
confident in drawing the general conclusion that the interactions
between hsp90 and Hop are substantially different than those between
hsp90 and PP5 or FKBP52. A second concern in interpreting the binding
data has to do with the effects of experimental conditions on the
results. Carello et al. (22) have previously shown that
deletion of the MEEVD sequence results in a loss of binding of Hop to
the immobilized C-terminal domain of hsp90. Chen et al. (23)
have previously demonstrated that mutation of this sequence to MAAVD in
chicken hsp90 resulted in a complete loss of binding to Hop immobilized
on an antibody resin and in a modest decrease in binding to FKBP52. We
obtained results identical to those of Carello et al. (22),
but when we performed the assay slightly differently, immobilizing Hop
by an N-terminal FLAG tag rather than immobilizing C90, we found that
mutating MEEVD to MAAVA had no effect on Hop binding. We also found
that this mutation inhibited binding to FKBP52 as well as to PP5, no matter which protein was immobilized. We interpret these results to
indicate that the MEEVD sequence is involved in binding to all three
TPR proteins but that multiple binding determinants exist for Hop. Only
under some experimental conditions does a single mutation result in a
complete loss of Hop binding.
The observation that there appear to be different binding determinants
for different TPR proteins, both within the TPR domain and within
hsp90, is somewhat surprising. It has been assumed that all TPR
proteins bind to the identical site on hsp90, and competition binding
studies have reinforced this notion (22, 24, 25). Such studies,
however, have also shown that bound TPR proteins can block the
interaction of hsp90 with cdc37, which presumably binds to a separate
nearby site (26, 27). If the TPR domains all bound to hsp90 in an
identical fashion, then biological differences between hsp90-binding
TPR proteins would reside outside their TPR domains. This hypothesis is
consistent with the observations that only Hop contains an
hsp70-binding domain, only FKBP52 contains a peptidylprolyl isomerase
domain, and only PP5 contains a phosphatase domain. However, the
situation may not be so simple. First, the TPR domains diverge
considerably in sequence. Second, the TPR domain of a large
immunophilin has the same biological activity as the full-length
protein in yeast, and inhibition of the peptidylprolyl isomerase
activity of a large immunophilin does not block its chaperone activity
in vitro (28-30). Thus, TPR domains may function as more
than simple targeting or docking domains. Even if the biological
differences between these TPR proteins prove to be mainly due to
interactions taking place outside their TPR domains, differences in
binding determinants may be exploited pharmacologically. Our results
suggest that there are structural differences between the binding sites
on hsp90 for PP5, FKBP52, and Hop. This, in turn, suggests that it
should be possible to develop inhibitors of specific TPR
complexes. These would have potential uses both as drugs and in the
study of proteins whose folding, processing, and subcellular
trafficking is dependent on particular TPR proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices that
form a right-handed superhelix (13) making it similar to the subunit of farnesyltransferase, which consists largely of a TPR domain
(14, 15). Both TPR domains contain a groove involved in protein-protein
interactions (13, 14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside for 5 h at
22 °C. Cells were harvested by centrifugation and stored frozen at
20 °C. The following procedures were all performed at 4 °C.
Frozen cell pellets were taken up in 30 ml of 20 mM Tris, 5 mM imidazole, 0.5 M NaCl, pH 8.0, containing
0.5 mg/ml lysozyme, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 20 mM benzamidine. After stirring for 30 min, and lysis by
sonication, lysates were centrifuged for 1 h at 18,000 × g. Streptomycin sulfate (final 2.5%) was added to the
clarified lysates, followed by stirring for 30 min. Samples were then
centrifuged at 18,000 × g for 30 min, and supernatants
were loaded onto a 5-ml HiTrap Chelating column (Amersham Pharmacia
Biotech). The His-tagged proteins were eluted using a gradient of
0.06-1 M imidazole in 20 mM Tris, pH 8.0, 500 mM NaCl. Fractions containing pure C90 protein, as assessed by SDS-PAGE, were pooled and dialyzed against 50 mM Hepes,
100 mM NaCl, pH 7.0. The concentrations of the wild-type
protein and of mutant 13 were initially determined both by amino acid
analysis and by dye binding (20). Because similar values (7.5%
difference) were obtained by both methods, dye binding was used to
determine the concentration of the remaining proteins, and values were
adjusted by 7.5% to correspond to those obtained by amino acid
analysis. Purified C90 proteins were stored at 80 °C.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the TPR domains of PP5, FKBP52,
and Hop. Sequences of the three repeats involved in binding to
hsp90 are shown for rat PP5, rabbit FKBP52, and murine Hop, the three
proteins used in the experiments presented here. Conserved residues
involved in PP5 binding to hsp90 are highlighted. Note that
Arg-74 of PP5, whose mutation only partially inhibits binding, is a Lys
residue in the other two proteins and that the three residues
absolutely required for binding are identical in all three
proteins.
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Fig. 2.
Effect of mutating a conserved Lys residue on
co-immunoprecipitation of hsp90 with PP5, FKBP52, and Hop. COS-7
cells were transfected with the indicated constructs, and the
FLAG-tagged proteins were immunoprecipitated with M2 antibody as
described under "Experimental Procedures." Residues mutated to Ala
were Lys-97 of PP5, Lys-354 of FKBP52, and Lys-429 of Hop.
Immunoprecipitates were analyzed by immunoblotting with an antibody to
hsp90 (top) or, as a control for expression of the
FLAG-tagged TPR proteins, with M2 antibody (bottom).
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Fig. 3.
Mutations in C90. The amino acid
sequence of C90, the TPR-binding domain of human hsp90- , is shown.
This domain extends from Met-628 to Asp-732. The locations of 13 mutants, in which individual acidic residues or groups of acidic
residues were mutated to Ala, are indicated.
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Fig. 4.
Binding of C90 mutants to immobilized TPR
proteins. The indicated FLAG-tagged TPR proteins were immobilized
on M2 beads as described under "Experimental Procedures," and the
indicated S-tagged C90 mutants were allowed to bind. After washing,
bound material was analyzed by SDS-PAGE and blotting with S-protein (to
detect S-tagged C90) or with antibody to the FLAG epitope (to detect
the indicated TPR proteins). wt, wild type.
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Fig. 5.
Binding of TPR proteins to immobilized C90
mutants. The indicated C90 mutants were immobilized on His·bind
beads as described under "Experimental Procedures," and the
indicated FLAG-tagged TPR proteins were allowed to bind. After washing,
bound material was analyzed by SDS-PAGE and immunoblotting with
antibody to the FLAG epitope (to detect the indicated TPR proteins) or
by blotting with S-protein (to detect S-tagged C90). wt,
wild type.
Summary of the effect of hsp90 mutations
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Fig. 6.
Effects of C90 mutations on chaperone
activity. Purified C90 (WT, wild type) and C90 mutants
were incubated at the indicated concentrations with rhodanese, and
their ability to block rhodanese aggregation was assessed as described
under "Experimental Procedures." Aggregation is shown as a function
of time in the absence of C90 (filled circles) or with C90
proteins present at ratios to rhodanese of 4:1 (open
circles), 8:1 (filled squares), or 20: 1 (open
squares).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix (13), a structure predicted to be found in C90.
We have now determined which of the 25 acidic residues in this
105-amino acid region are required for binding to the TPR domains of
PP5, FKBP52, and Hop; all three proteins bind to C90 and contain the
same conserved basic residues. Our results confirm and extend previous
data suggesting that the MEEVD sequence at the C terminus of hsp90 was
important for binding TPR proteins and that different TPR proteins
competed for binding to the same site (22, 23). The data presented
here, however, suggest a more complex picture. Although it seems clear
that the MEEVD sequence is involved in binding to all three TPR
proteins, there are also differences in their binding determinants. A
cluster of four acidic amino acids adjacent to the MEEVD sequence
(Glu-720, Asp-722, Asp-723, and Asp-724) is involved in binding to
FKBP52 and PP5, proteins that are found in mature hsp90 complexes. Its mutation does not affect the binding of Hop, however, which is found in
intermediate complexes. Mutation of either of these C-terminal sequences, while inhibiting TPR binding, does not affect the chaperone activity of C90, suggesting that the site of chaperone activity is
located more toward the N terminus of this domain. Mutation of Glu-651
and Asp-653 decreases not only chaperone activity but Hop binding.
Thus, the site of Hop binding, but not that of FKBP52 or PP5 binding,
appears to overlap with the site of chaperone activity. One might
speculate that Hop enters the hsp90 complex at an intermediate stage of
protein folding because it is recognized by hsp90 as a misfolded
protein, binding to one of the two available chaperone sites. Hop
differs from FKBP52 and PP5 in another respect; a Lys residue conserved
among these three proteins is absolutely required for binding of PP5 or
FKBP52 to hsp90 but not for binding of Hop. This suggests that there
are differences between the interactions between Hop and hsp90 and
those between FKBP52 or PP5 and hsp90. This could either involve a
difference in the binding of the TPR domain of Hop itself or simply the
presence of additional binding determinants outside the TPR domain.
With the exception of Hop, this conserved Lys residue may be of general
importance in the binding of TPR proteins to hsp90. Recently, several
investigators have found that its mutation prevents binding of hsp90 to
CyP-402,
FKBP51,3 and
CNS1.4
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ACKNOWLEDGEMENTS |
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We thank Drs. E. Baulieu and M. Lässle for gifts of FKBP52 and Hop cDNA; Dr. L. Dangott (Texas A & M University) for performing amino acid analysis; and Dr. J. Scammell for careful reading of the manuscript.
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Note Added in Proof |
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It has now been shown that sequence alignments were indeed misleading and that Lys-301 rather than Lys-429 of Hop corresponds to Lys-97 of PP5 (Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F. U., and Moarefi, I. (2000) Cell 101, 199-210).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL 47063 and DK 55877 (to M. C.).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. Tel.: 334-460-6782;
Fax: 334-460-6798; E-mail: michaelc@jaguar1.usouthal.edu.
Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M001625200
2 T. Ratajczak, personal communication.
3 J. Scammell, personal communication.
4 R. Gaber, personal communication.
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ABBREVIATIONS |
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The abbreviations used are: hsp90, heat shock protein 90; TPR, tetratricopeptide repeat; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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