From the S. C. Johnson Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259
Received for publication, January 29, 2003
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ABSTRACT |
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Hsp90 assembles with steroid receptors and
other client proteins in association with one or more Hsp90-binding
cochaperones, some of which contain a common tetratricopeptide repeat
(TPR) domain. Included in the TPR cochaperones are the
Hsp70-Hsp90-organizing protein Hop, the FK506-binding immunophilins
FKBP52 and FKBP51, the cyclosporin A-binding immunophilin CyP40, and
protein phosphatase PP5. The TPR domains from these proteins have
similar x-ray crystallographic structures and target cochaperone
binding to the MEEVD sequence that terminates Hsp90. However, despite
these similarities, the TPR cochaperones have distinctive properties
for binding Hsp90 and assembling with Hsp90·steroid receptor
complexes. To identify structural features that differentiate binding
of FKBP51 and FKBP52 to Hsp90, we generated an assortment of truncation
mutants and chimeras that were compared for coimmunoprecipitation with
Hsp90. Although the core TPR domain (approximately amino acids
260-400) of FKBP51 and FKBP52 is required for Hsp90 binding, the
C-terminal 60 amino acids (~400-end) also influence Hsp90 binding.
More specifically, we find that amino acids 400-420 play a critical
role for Hsp90 binding by either FKBP. Within this 20-amino acid
region, we have identified a consensus sequence motif that is also
present in some other TPR cochaperones. Additionally, the final 30 amino acids of FKBP51 enhance binding to Hsp90, whereas the
corresponding region of FKBP52 moderates binding to Hsp90. Taking into
account the x-ray crystal structure for FKBP51, we conclude that the
C-terminal regions of FKBP51 and FKBP52 outside the core TPR domains
are likely to assume alternative conformations that significantly impact Hsp90 binding.
Hsp90, typically the most abundant cytoplasmic chaperone in
vertebrate cells, serves a vital role in cellular signaling by regulating the folding, activity, and stability of a wide range of
client proteins, as exemplified by steroid receptors and protein kinases (1). Client protein complexes contain not only Hsp90 but also
one or more cochaperones that partner with Hsp90. One class of
Hsp90-binding cochaperone is composed of proteins with a characteristic
tetratricopeptide repeat
(TPR)1 domain that forms an
Hsp90 binding site (2). Among the TPR cochaperones of Hsp90 are
Hop/Sti1, an adaptor chaperone that also binds Hsp70 (3, 4), protein
phosphatase PP5 (5), and members of both the FK506- and cyclosporin
A-binding families of immunophilins (6-9). The TPR cochaperones
compete for binding the C-terminal region of Hsp90 (10-12), and the
highly conserved MEEVD sequence that terminates eukaryotic Hsp90 is a
common target for TPR interactions (13-16). Mutation of the MEEVD
sequence typically inhibits binding by a TPR cochaperone, although
mutations in other C-terminal sequences impact binding by TPR
cochaperones differentially (13, 17).
The immunophilin-related TPR cochaperones contain peptidylprolyl
isomerase domains that also serve as the binding site for immunosuppressive drugs, yet immunophilins can have differential effects on the function of Hsp90 client proteins. In particular, FKBP52
and FKBP51, which share ~70% amino acid sequence similarity, affect
hormone binding by the glucocorticoid receptor (GR) in opposing
manners. A study in the yeast Saccharomyces cerevisiae, which lacks endogenous counterparts to FKBP52 or FKBP51, has shown that
FKBP52, specifically, can elevate GR responsiveness to hormone (18).
Although FKBP51 alone does not reduce GR activity in yeast, it can
effectively block the potentiation mediated by FKBP52 when coexpressed
(18). Scammell and his colleagues (19-21) have shown that cortisol
insensitivity observed in New World primates is facilitated by a
constitutive overexpression of FKBP51, suggesting a physiological
relevance for FKBP interactions with GR·Hsp90 complexes. Bourgeois
and colleagues (22) first noted that the gene for FKBP51 is
up-regulated by glucocorticoids, and this observation has been
confirmed repeatedly by recent gene expression profiles of
steroid-responsive genes. The inducibility of FKBP51 by glucocorticoids could provide a mechanism for partial desensitization of cells subsequent to an initial exposure to hormone (23).
It is not clear which structural features are responsible for the
differential effects of FKBP52 and FKBP51. Three-dimensional crystallographic structures for FKBP51 have recently been solved (24),
but there is no corresponding structure for FKBP52. Based on the 70%
amino acid sequence similarity of FKBP51 and FKBP52 and the
conservation of apparent domain sequences, there is no reason to
suspect a dramatic structural difference. Nonetheless, previous studies
from us showed that mutations in the C-terminal half of Hsp90 have
different effects on binding by FKBP52 versus FKBP51 (13).
Also, we observed that an exchange of sequences between the FKBP TPR
domains had no effect on Hsp90 binding by FKBP52 but blocked binding by
FKBP51 (25). These observations suggested that each FKBP has a
distinctive interaction with Hsp90. In the current study, we have taken
advantage of the x-ray crystallographic structure of FKBP51 to map
functionally sequences of FKBP51 and FKBP52 that are important for
Hsp90 binding. We find that sequences in the C-terminal region, both
inside and outside the TPR domain, greatly influence FKBP binding to Hsp90.
Preparation of Mutant cDNAs--
In vitro
expression plasmids containing either human FKBP51 or FKBP52 cDNA
inserted into pSPUTK (Stratagene, La Jolla, CA) were used to generate
mutants used in these studies. To construct the C-terminal truncations,
stop codons were introduced by site-directed mutagenesis (QuikChange
kit, Stratagene). To help clarify our naming convention for many of the
mutants generated for this study, it is helpful to note that FKBP52,
relative to FKBP51, contains a two-amino acid insert in the loop
connecting FK1 and FK2 domains; thus, the position of corresponding
mutations in the C-terminal halves of either FKBP will differ by two
residues. For example, the FKBP51 truncation mutant N404 contains an
engineered stop codon at position 405; the equivalent FKBP52 truncation
mutant, N406, contains a stop codon at position 407. The FK mutants
were generated by introducing stop codons at position 258 or 260, respectively, which lies in the linker region between FK2 and the TPR
domain. The TPR mutants were created by first introducing an F252M or F254M substitution and then removing all upstream coding sequences. FKBP chimeric cDNAs were constructed by two different approaches. The FKBP51-395 (positions 1-395 code for FKBP51 and positions 396-end
code for FKBP52) and the converse FKBP52-397 chimeric cDNA were
originally cloned into yeast expression vectors (18). These cDNA
were PCR amplified from yeast vectors and subcloned into pSPUTK. Other
chimeras were created as follows. A gene fragment encoding the
N-terminal portion of the desired chimeric protein was generated by
PCR. A second gene fragment encoding the C-terminal portion of the
desired chimera was separately generated by PCR. Primers for the two
fragments were designed such that sequences surrounding the desired
fusion site were complementary. The resulting DNA products were gel
purified and used as megaprimers in a reaction with the appropriate 5'-
and 3'-primers to generate the full-length chimeric cDNA. The final
PCR product was subcloned into pSPUTK. Sequence changes in mutated
cDNAs and all PCR-generated products were confirmed by automated
DNA sequencing.
In Vitro Hsp90 Binding and PR Complex Assembly--
The
Hsp90 and PR binding abilities of wild type proteins and mutants were
analyzed by a coimmunoprecipitation approach as described previously
(25). Briefly, radiolabeled immunophilins were synthesized from plasmid
DNA templates in an in vitro transcription/translation system (TNT lysate, Promega). Radiolabeled products were
quantitated by densitometry of gel autoradiographs, and an equimolar
amount of each radiolabeled product was added separately to 100 µl of rabbit reticulocyte lysate (RL; Green Hectares, Oregon, WI). Each RL
sample was added to a 10-µl pellet of protein G-Sepharose (Amersham Biosciences) prebound with 10 µg of H90-10, a specific anti-Hsp90 mouse monoclonal antibody. After incubation on ice for 1 h,
unbound proteins were removed by washing three times in 1 ml of wash
buffer (20 mM Tris-HCl, pH 7.4, 50 mM KCl, 1%
Tween 20). Bound proteins were then extracted with SDS sample buffer
and separated by SDS-PAGE. The assembly of PR complexes was analyzed in
a similar manner, except 200 µl of RL was supplemented with an
ATP-regenerating system, samples were incubated with 1 µg of
recombinant PR bound to PR22-protein A-Sepharose, and incubations were
at 30 °C for 30 min. After electrophoresis, gels were stained with
Coomassie Brilliant Blue to visualize total proteins. Gels were then
dried and autoradiographed to visualize radiolabeled proteins, and
bands were quantitated by densitometry using a Fluor-S Imager
(Bio-Rad).
Yeast Strains, Plasmids, and Methods--
FKBP functional assays
were performed in S. cerevisiae essentially as described
previously (18). All experiments were performed using a GR reporter
strain in the W303a background (MATa
leu2-112 ura3-1 trp1-1
his3-11, 14 ade2-1 can1-100 GAL
SUC2). Parental cells were transformed with a plasmid that
constitutively expresses rat GR, a second plasmid that expresses
Hormone induction assays were performed as described previously (18).
Briefly, yeast strains were grown in selective medium at 25 °C, and
the absorbance at 600 nm (A600) of the
culture was monitored to ensure exponential growth. Reporter gene
expression was induced by adding deoxycorticosterone (25 nM
final concentration) to log phase cultures. Starting 70 min later,
cells were sampled at 10-min intervals over the next 40 min for
reporter activity. The three-dimensional crystallographic structure for FKBP51
(Protein Data Bank code 1KT1) depicted in Fig.
1A reveals three major
structural domains. Of the two FKBP12-like domains, peptidylprolyl isomerase and FK506 binding activities reside in FK1 (24). The Hsp90 binding TPR domain has a structure similar to that of other described TPR domains, in particular the Hsp90 binding domains from PP5
(26), Hop (16), and CyP40 (27). The consensus TPR motifs terminate with
helix 6 (H6, Fig. 1B); however, similar to structures for
PP5 and CyP40, there is a seventh helix that extends beyond the core
TPR domain for a minimum of 23 amino acids. How much further H7 may
extend is unclear because the final 36 amino acids could not be
resolved from crystal data. Although a crystal structure for FKBP52 has
not been reported, it seems likely that it will share the same
overall domain organization as FKBP51.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase from a glucocorticoid-regulated promoter, and a third
plasmid constitutively expressing one of the human immunophilins. Cells
were grown in minimal medium containing 0.67% (w/v) yeast nitrogen
base without amino acids, 2% (w/v) glucose, the appropriate SC
supplement mixture (Q-biogene, Carlsbad, CA); for growth on plates the
culture medium was supplemented with 1.6% w/v agar.
-Galactosidase activity was measured by adding
100 µl of culture to an equal volume of Gal-Screen assay solution
(Tropix, Bedford, MA) according to the manufacturer's instructions.
Reporter expression rate was calculated as the linear slope of relative
light units versus A600/1,000. For
each strain tested, hormone-induced reporter expression rate was
determined with two to four independent transformants to assure
consistency of results.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
FKBP51 structure. A,
FKBP51 contains two FKBP12-like domains. FK1 has peptidylprolyl
isomerase activity and provides the binding site for
immunosuppressive drugs; FK2 has a similar fold but differs at several
amino acid positions important for peptidylprolyl isomerase
activity and drug binding. The TPR domain forms an Hsp90 binding site
and is structurally similar to other Hsp90 binding TPR domains. For
each domain, the putative or known binding site is indicated by label
positions. The N-terminal 27 amino acids and C-terminal 36 amino acids
of full-length FKBP51 were unresolved in this structure. B,
the three repeat motifs in the TPR domain form six -helices
(H1-H6). A seventh helix has a core region that forms interactions
with H6 and a region that extends beyond the TPR domain. The complete
extent of H7 is unknown because the observed structure terminates at
position 421 of 457. The locations of Lys-352 and Arg-404 are
indicated; these positions are relevant to mutations described in later
figures.
FKBP Regions Required for Hsp90 and Receptor Interactions--
As
presented in Fig. 2, an initial set of
FKBP51 and FKBP52 mutants (Fig. 2A) was surveyed to compare
interactions with Hsp90 and steroid receptor complexes.
Coimmunoprecipitations of radiolabeled FKBP51 mutants (Fig.
2B) and FKBP52 mutants (Fig. 2C) were used to
monitor Hsp90 binding (left panels) and assembly into PR
complexes (right panels). We consistently found that
incorporation of FKBP51 exceeded that of FKBP52 by 2-3-fold in Hsp90
complexes (compare lanes 1 and 2 in each data
set). In line with previous observations (25), FKBP51 recovery in PR
complexes exceeded the recovery of FKBP52 by 5-fold or greater (compare
lanes 8 and 9 in each set). The TPR truncation
mutants, which included the TPR domain plus C-terminal sequences, were
sufficient for binding Hsp90 (lane 3) and assembling with PR
complexes (lane 10). The TPR region was necessary for
binding Hsp90 and assembling with PR because the FK domains showed no
interactions (lanes 4 and 11 in each set). The
importance of the TPR domain is demonstrated further by point mutation
at one of the carboxylate clamp residues (K352A for FKBP51 and K354A
for FKBP52) that abrogates Hsp90 binding and PR association
(lanes 5 and 12). To test whether C-terminal sequences influence protein interactions, truncation mutants (N404 and
N406) were generated which lacked sequences within H7 extension (see
Fig. 1B) and beyond. Despite retention of the core TPR
domain in these constructs, Hsp90 binding (lane 6 in both
sets) and PR association (lane 13 in both sets) were largely
abrogated. The final samples in this mutant series were a pair of
chimeric constructs (395 for FKBP51 and 397 for FKBP52) in which the
region from the H7 extension through the C terminus was exchanged
between FKBPs. This exchange had little effect on FKBP52 (Fig.
2C, lanes 7 and 14) but greatly
diminished interactions of FKBP51 with Hsp90 and PR (Fig. 2B,
lanes 7 and 14). In an earlier study (25) in which the
C-terminal region was retained but TPR motifs were exchanged, we
observed a similar phenomenon; the FKBP52-based construct functioned normally, but the FKBP51-based construct lost the ability to bind Hsp90
or assemble with PR complexes.
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Taking advantage of the yeast model for FKBP52-dependent
potentiation of GR signaling (18), we further tested FKBP52 constructs for function in vivo (Fig. 2D). First, note that
FKBP52 significantly enhances hormone-induced -galactosidase
activity compared with yeast expressing FKBP51 or lacking either FKBP
(Vector). None of the FKBP52 mutants displayed potentiation
of GR signaling except the C-terminal chimera 397. This is consistent
with the dual requirement for Hsp90 binding and FK1 peptidylprolyl
isomerase activity for FKBP52 to elevate GR hormone binding
affinity (18). Because 397 retains wild type activity in this assay, it
appears that specific sequences in the tail region of FKBP52 are not
critical for its function in GR potentiation.
Hsp90 Binding by C-terminal Truncation Mutants--
The
defect in Hsp90 interactions apparent with FKBP51-N404 and FKBP52-N406
was unexpected and raised the question of which sequences downstream
from the core TPR domain are minimally required for Hsp90 binding. To
begin addressing this question, a series of FKBP truncation mutants was
generated which focused on this region, and these were tested for
coimmunoprecipitation with Hsp90. Densitometric measurements were taken
from gel autoradiographs similar to those in Fig. 2, and these data
were plotted as shown in Fig. 3.
Significant recovery of FKBP51 mutants in Hsp90 complexes (solid
circles) began with constructs that included residues 415-420, near the downstream end of H7. Full Hsp90 binding was only observed with constructs containing sequences beyond 430.
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Truncations of FKBP52 (open circles) differed from FKBP51 mutants in two ways. First, note that there is a leftward shift in the Hsp90 binding curve for the FKBP52 series compared with the FKBP51 series. Thus minimally sized FKBP52 truncations that retain measurable levels of Hsp90 binding are 5-8 amino acids shorter than the smallest FKBP51 constructs that retain Hsp90 binding. The second difference pertains to the influence of sequences beyond position 430. Whereas the C-terminal region heightened Hsp90 binding by FKBP51, there appears to be a modest inhibition of Hsp90 binding by the corresponding region of FKBP52. Mutants terminating at approximately position 430 bind Hsp90 equally. However, the inclusion of sequences beyond this point moderated binding of FKBP52 to Hsp90 but enhanced binding by FKBP51.
Hsp90 Binding by FKBP51 Chimeras Containing C-terminal Portions of
FKBP52--
In marked contrast to the corresponding FKBP52 chimeras,
swapping either the TPR domain (25) or the adjacent C-terminal tail
(chimera 395 in Fig. 2B) abrogated FKBP51 binding to Hsp90. Swapping both regions together had no effect on Hsp90 binding. These
observations suggest important intramolecular interactions between the
C-terminal tail region and the TPR domain which are distinctive in
FKBP51 and FKBP52. As shown in Fig. 4,
additional FKBP51 chimeras focusing on the TPR domain boundary from H5
through H7 were constructed to probe more thoroughly for potential
interactions. Comparing hydrophobicity patterns in this region from
FKBP51 and FKBP52, the two proteins are similar (Fig. 4A),
although there is only 50% amino acid sequence identity. Another
series of FKBP51 chimeras was generated in which an exchange occurred
at one of eight positions along H6, H7, or just beyond H7 (seven of
these sites are indicated in Fig. 4A). Radiolabeled products
were synthesized and compared for coimmunoprecipitation with Hsp90
(Fig. 4B). As observed consistently, recovery of wild type
FKBP51 in Hsp90 complexes exceeded that of wild type FKBP52 (compare
first two lanes). Chimeras containing the H6 region and
beyond from FKBP52 (365, 374, and 378) retained Hsp90 binding. When the
exchange occurred within H7 (395, 404, and 413), there was a complete
loss of Hsp90 binding. Hsp90 binding was again observed when the
exchange was restricted to sequences beyond 422. To reiterate, we
observed no defect in Hsp90 binding by FKBP52 chimeras that contained
similar tail regions from FKBP51 (for example, for results with FKBP52
chimera 397, see Fig. 2C). Collectively, these observations
suggest that there is an interaction within the region from H6 through
H7 extended which is uniquely required by FKBP51 for Hsp90 binding.
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An additional analysis was undertaken in which a series of C-terminal truncation mutants was generated from the defective chimera FKBP51-395. Similar to previous binding assays, truncations of 395 were examined for coimmunoprecipitation with Hsp90 on gel autoradiographs. The quantitated data were plotted in Fig. 4C (solid triangles). Also plotted in this figure are the quantitated data for FKBP51 chimeras (solid circles) that were examined in Fig. 4B and for FKBP52 and the FKBP52-397 chimera (open circles). Consistent with the behavior of FKBP52 truncation mutants (Fig. 3), the FKBP52-397 chimera displayed greater binding than wild type FKBP52 (compare open circles) presumably because of the loss of inhibitory sequences beyond position 430 of FKBP52. As seen from the gel data in Fig. 4B, the FKBP51 chimeras (solid circles) had a pattern of loss then recovery of Hsp90 binding as the fusion point in chimeras progressed from H6 through H7. Interestingly, truncations of FKBP51-395 (triangles) showed that Hsp90 binding could be partially restored in constructs that terminate between 410 and 430. The sharp boundary at ~410 corresponds well with the Hsp90 binding boundary observed with FKBP52 truncation. Likewise, the trailing boundary near 430 corresponds to the inhibition boundary deduced from FKBP52 truncation mutants.
Functional Analysis of FKBP52 Sequences in the 400-420 Region of
H7--
Experimental results with FKBP52 and FKBP51 mutants point to
the extended portion of H7 (amino acids 400-420) as being important for Hsp90 binding and FKBP function. This region of FKBP52 was analyzed
further, as shown in Fig. 5. Alignment of
FKBP sequences in this region highlights the conservation of amino
acids 406-415 (Fig. 5A, FKBP52 numbering), a 10-amino acid
stretch consisting of a highly charged segment followed by YANMF. A
series of FKBP52 truncation mutants that target this region was
generated and tested for Hsp90 binding and assembly with PR complexes
(Fig. 5B). As seen previously (Fig. 2), binding to Hsp90 and
assembly into receptor complexes were greatly reduced with N406, but
both interactions were restored when the C terminus was extended to 414 and beyond. Yeast GR reporter strains were generated to correlate
protein-protein interactions in vitro with FKBP52 function
in vivo (Fig. 5C). Corresponding with weak Hsp90
and PR interactions, N406 and N410 lacked the ability to potentiate GR
signaling; however, potentiation was significantly boosted with N414
and larger mutants in parallel with Hsp90 and PR interaction
patterns.
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Sequence data bases were accessed to determine whether other
TPR-dependent Hsp90 cochaperones share a motif similar to
the 406-415 segment. For each cochaperone we selected sequences that lie in the same relative downstream juxtaposition to TPR motifs. These
juxta-TPR sequences are aligned in Fig.
6. Included in the comparison are the
Hsp90-binding FKBP family members FKBP52, FKBP51, FKBP36 (28), and
Xap2/AIP (29, 30). CyP40 and PP5 sequences are present, as well as
sequences from Hop (31) and CHIP (32). As shown above the alignment, an
11-amino acid motif, what we term the charge-Y motif, was identified
with the consensus organization +
+X
YXXMF,
where
represents Glu or Asp, + represents Lys or Arg,
represents a hydrophobic amino acid, and X represents any amino acid. There is also a negatively charged amino acid 5 positions further downstream which may relate to the consensus. Six of the nine
Hsp90 cochaperones match the consensus in at least four of nine
positions (indicated by left arrowhead).
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DISCUSSION |
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FKBP52 and FKBP51 are closely related Hsp90-binding cochaperones, yet they have distinctive patterns of interaction with Hsp90 and Hsp90 client proteins such as steroid receptors. In an effort to understand better the structural basis for distinctive interactions with Hsp90, we performed a mutagenic analysis of sequences in FKBP51 and FKBP52 which impact Hsp90 binding. Both immunophilins have a core TPR domain that is necessary for binding to Hsp90, but this core domain is not sufficient for full binding to Hsp90 (Fig. 2). We have identified a conserved region, the charge-Y motif, which lies immediately downstream from the TPR domain and is required for Hsp90 binding (Figs. 3 and 5). A similar sequence is found in some other Hsp90-binding TPR proteins (Fig. 6). Further downstream in either FKBP, unique sequences within the final 30 amino acids appear to distinguish the relatively higher Hsp90 binding affinity of FKBP51 compared with FBP52 (Figs. 3 and 4). The FKBPs are distinguished further by an intramolecular interaction peculiar to FKBP51 which involves H7, the final helix in the core TPR domain, and the adjacent charge-Y motif (Fig. 4). Thus, the Hsp90 binding properties of these two TPR cochaperones result from a combination of the core TPR domain and the influence of C-terminal sequences outside this domain. Consistent with the notion of alternative modes of interaction with Hsp90, point mutations in the C-terminal region of Hsp90 have distinct effects on the binding of individual TPR cochaperones (13, 17). This suggests that cochaperones might interface with distinct structural features of Hsp90 in addition to a common interaction with the C-terminal MEEVD.
Jackknife Model for Charge-Y Motif Participation in Hsp90
Binding--
According to the crystal structure for FKBP51 (Fig. 1),
the charge-Y motif lies in a portion of H7 that extends beyond the core
TPR domain. As depicted in Fig. 7, we
hypothesize two alternative conformational states of H7 sequences that
could account for our mutagenic data. In the first state (Fig.
7A), H7 exists in the extended conformation that is
consistent with the FKBP51 crystal structure. Here, charge-Y could
directly contact Hsp90 and complement or facilitate binding through the
core TPR domain. However, it is difficult to reconcile this
conformational state with data from FKBP51 chimeras which argue for
matching of sequences between the core and extended portions of H7
(Figs. 2 and 4). Yet based on the FKBP51 crystal structure, there is
unlikely to be direct contact between these regions. We propose the
alternative possibility (Fig. 7B) that H7 may be disrupted
in some circumstances, forming an eighth helix that continues the
anti-parallel pattern of interactions observed with H1 through H7. In
this conformational state, the putative H8 may contribute to core TPR
domain interactions that enhance TPR affinity for Hsp90.
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Although an anti-parallel H8 is not observed in the static FKBP51
crystal structure, crystal packing may not have favored a conformation
of FKBP51 that exists in solution or that is induced by binding to
Hsp90. Furthermore, there are precedents for alternative conformational
states in TPR domains such as proposed in Fig. 7. Walkinshaw and
colleagues (27) obtained two crystal forms for CyP40 from which two TPR
conformations were resolved. One structure contained a TPR domain
similar to PP5, Hop-TPR2a, and FKBP51. In the alternate structure, the
loops separating H2-H3 and H3-H4 have been reorganized into -helical
forms, thus resulting in a single, greatly extended H2. A similar
phenomenon was observed in the crystal structures for peroxisomal TPR
protein PEX5. The structure for human PEX5 has the canonical
anti-parallel helix arrangement (33). In contrast to this structure,
Kumar et al. (34) obtained a structure for trypanosome PEX5
in which H5 and H6 were fused into a single extended helix. They
proposed the possibility that certain TPR domains may naturally assume
alternative conformations through extension versus folding
back of helices, what they termed the "jackknife model" for TPR
motif rearrangement (34). The alternate FKBP structures proposed in
Fig. 7 are analogous to the open and closed states of the TPR jackknife
model, except in this case the sequences involved are not within the
consensus TPR motifs that form H1 through H6.
Chimeric data suggest that FKBP51 may assume the closed conformation (Fig. 7B), whereas FKBP52 binds Hsp90 in an open conformation (Fig. 7A). One can visualize in the closed conformation how the charge-Y motif and adjacent amino acids may interact with amino acids in the core portion of H7 or even with side chains extending from H6. The C-terminal 30-35 amino acids that are unresolved in the FKBP51 crystal structure are not illustrated in Fig. 7. However, this region also impacts Hsp90 binding (Figs. 3 and 4), enhancing binding by FKBP51 and moderating binding by FKBP52. In the jackknife model, this tail region would have to undergo a dramatic positional swing, but such a difference perhaps contributes to influences of the tail on Hsp90 binding.
Further structural studies are needed to test the jackknife model, and alternative explanations for our observations remain viable. For example, the charge-Y motif lies within a region that is purported to be a calmodulin binding site in FKBP52 (35). We considered whether Ca2+/calmodulin interactions could either influence the conformational state of this region or provide an alternative mechanism for distinguishing FKBP51 and FKBP52 interactions with Hsp90. However, we think a role for calmodulin is unlikely for several reasons. First, the putative calmodulin binding motif scores very weakly for FKBP52 and FKBP51 when analyzed in the Calmodulin Target Data base (calcium.uhnres.utoronto.ca/ctdb/flash.htm). Second, we have never observed calmodulin in FKBP complexes with Hsp90 or steroid receptors. Finally, neither Ca2+ nor the Ca2+ chelators EGTA or EDTA alter FKBP binding to Hsp90 (results not shown).
General Significance of TPR Cochaperone Interactions with Hsp90 and
Client Proteins--
Hsp90 serves a large number of client proteins
that regulate cellular pathways, and in every case examined one or more
cochaperones accompanied Hsp90 in client protein complexes. Individual
clients displayed distinct preferences for certain Hsp90 cochaperones. For example, among the TPR cochaperones, Xap2/AIP is found in arylhydrocarbon receptor complexes but not steroid receptor or kinase
complexes (29, 30). FKBP51, FKBP52, PP5, and CyP40 associate
differentially with progesterone, estrogen, and glucocorticoid receptor
complexes in a receptor-specific manner (25, 36, 37). Preferential
assembly with a client may be caused by direct interactions between
client and the Hsp90 cochaperone (24), but the cochaperone may also
influence how Hsp90 interfaces with client, either stabilizing or
destabilizing client interactions relative to other Hsp90/cochaperone
pairs. Thus, the TPR cochaperones that competitively interact with the
MEEVD terminus of Hsp90 might, through unique contacts with Hsp90,
induce distinct structural/functional changes in Hsp90 which elaborate
the chaperoning of client proteins.
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ACKNOWLEDGEMENT |
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We thank Dr. Cindy Sinars for helpful discussions on immunophilin structure.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant DK48218 (to D. F. S.) and the Mayo Foundation.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 Biochemistry
and Molecular Biology, Mayo Clinic Scottsdale, 13400 E. Shea Blvd.,
Scottsdale, AZ 85259. Tel.: 480-301-6595; Fax: 480-301-3384; E-mail: smith.david26@mayo.edu.
Published, JBC Papers in Press, February 27, 2003, DOI 10.1074/jbc.M300955200
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ABBREVIATIONS |
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The abbreviations used are: TPR, tetratricopeptide repeat; CyP40, cyclosporin A-binding immunophilin; FKBP, FK506-binding protein; GR, glucocorticoid receptor; PR, progesterone receptor; Hop, Hsp70-Hsp90-organizing protein; PP, protein phosphatase; RL, reticulocyte lysate.
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1. | Richter, K., and Buchner, J. (2001) J. Cell. Physiol. 188, 281-290[CrossRef][Medline] [Order article via Infotrieve] |
2. | Blatch, G. L., and Lassle, M. (1999) Bioessays 21, 932-939[CrossRef][Medline] [Order article via Infotrieve] |
3. | Chen, S., Prapapanich, V., Rimerman, R. A., Honore, B., and Smith, D. F. (1996) Mol. Endocrinol. 10, 682-693[Abstract] |
4. |
Lassle, M.,
Blatch, G. L.,
Kundra, V.,
Takatori, T.,
and Zetter, B. R.
(1997)
J. Biol. Chem.
272,
1876-1884 |
5. |
Chen, M. S.,
Silverstein, A. M.,
Pratt, W. B.,
and Chinkers, M.
(1996)
J. Biol. Chem.
271,
32315-32320 |
6. | Tai, P. K., Albers, M. W., Chang, H., Faber, L. E., and Schreiber, S. L. (1992) Science 256, 1315-1318[Medline] [Order article via Infotrieve] |
7. | Peattie, D. A., Harding, M. W., Fleming, M. A., DeCenzo, M. T., Lippke, J. A., Livingston, D. J., and Benasutti, M. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10974-10978[Abstract] |
8. |
Ratajczak, T.,
Carrello, A.,
Mark, P. J.,
Warner, B. J.,
Simpson, R. J.,
Moritz, R. L.,
and House, A. K.
(1993)
J. Biol. Chem.
268,
13187-13192 |
9. |
Radanyi, C.,
Chambraud, B.,
and Baulieu, E.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11197-11201 |
10. |
Owens-Grillo, J.,
Hoffmann, K.,
Hutchison, K.,
Yem, A.,
Deibel, M.,
Handschumacher, R.,
and Pratt, W.
(1995)
J. Biol. Chem.
270,
20479-20484 |
11. |
Ratajczak, T.,
and Carrello, A.
(1996)
J. Biol. Chem.
271,
2961-2965 |
12. | Nair, S. C., Rimerman, R. A., Toran, E. J., Chen, S., Prapapanich, V., Butts, R. N., and Smith, D. F. (1997) Mol. Cell. Biol. 17, 594-603[Abstract] |
13. | Chen, S., Sullivan, W. P., Toft, D. O., and Smith, D. F. (1998) Cell Stress Chaperones 3, 118-129[Medline] [Order article via Infotrieve] |
14. |
Carrello, A.,
Ingley, E.,
Minchin, R. F.,
Tsai, S.,
and Ratajczak, T.
(1999)
J. Biol. Chem.
274,
2682-2689 |
15. |
Russell, L. C.,
Whitt, S. R.,
Chen, M. S.,
and Chinkers, M.
(1999)
J. Biol. Chem.
274,
20060-20063 |
16. | Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder, L., Bartunik, H., Hartl, F. U., and Moarefi, I. (2000) Cell 101, 199-210[Medline] [Order article via Infotrieve] |
17. |
Ramsey, A. J.,
Russell, L. C.,
Whitt, S. R.,
and Chinkers, M.
(2000)
J. Biol. Chem.
275,
17857-17862 |
18. |
Riggs, D. L.,
Roberts, P. J.,
Chirillo, S. C.,
Cheung-Flynn, J.,
Prapapanich, V.,
Ratajczak, T.,
Gaber, R.,
Picard, D.,
and Smith, D. F.
(2003)
EMBO J.
22,
1158-1167 |
19. |
Reynolds, P. D.,
Ruan, Y.,
Smith, D. F.,
and Scammell, J. G.
(1999)
J. Clin. Endocrinol. Metab.
84,
663-669 |
20. |
Denny, W. B.,
Valentine, D. L.,
Reynolds, P. D.,
Smith, D. F.,
and Scammell, J. G.
(2000)
Endocrinology
141,
4107-4113 |
21. | Scammell, J. G., Denny, W. B., Valentine, D. L., and Smith, D. F. (2001) Gen. Comp. Endocrinol. 124, 152-165[CrossRef][Medline] [Order article via Infotrieve] |
22. | Baughman, G., Wiederrecht, G. J., Campbell, N. F., Martin, M. M., and Bourgeois, S. (1995) Mol. Cell. Biol. 15, 4395-4402[Abstract] |
23. |
Cheung, J.,
and Smith, D. F.
(2000)
Mol. Endocrinol.
14,
939-946 |
24. |
Sinars, C. R.,
Cheung-Flynn, J.,
Rimerman, R. A.,
Scammell, J. G.,
Smith, D. F.,
and Clardy, J.
(2003)
Proc. Natl. Acad. Sci. U. S. A.
100,
868-873 |
25. |
Barent, R. L.,
Nair, S. C.,
Carr, D. C.,
Ruan, Y.,
Rimerman, R. A.,
Fulton, J.,
Zhang, Y.,
and Smith, D. F.
(1998)
Mol. Endocrinol.
12,
342-354 |
26. |
Das, A. K.,
Cohen, P. W.,
and Barford, D.
(1998)
EMBO J.
17,
1192-1199 |
27. | Taylor, P., Dornan, J., Carrello, A., Minchin, R. F., Ratajczak, T., and Walkinshaw, M. D. (2001) Structure 9, 431-438[CrossRef][Medline] [Order article via Infotrieve] |
28. | Meng, X., Lu, X., Morris, C. A., and Keating, M. T. (1998) Genomics 52, 130-137[CrossRef][Medline] [Order article via Infotrieve] |
29. |
Ma, Q.,
and Whitlock, J. P., Jr.
(1997)
J. Biol. Chem.
272,
8878-8884 |
30. |
Meyer, B. K.,
Pray-Grant, M. G.,
Vanden Heuvel, J. P.,
and Perdew, G. H.
(1998)
Mol. Cell. Biol.
18,
978-988 |
31. |
Honore, B.,
Leffers, H.,
Madsen, P.,
Rasmussen, H. H.,
Vandekerckhove, J.,
and Celis, J. E.
(1992)
J. Biol. Chem.
267,
8485-8491 |
32. |
Ballinger, C. A.,
Connell, P.,
Wu, Y.,
Hu, Z.,
Thompson, L. J.,
Yin, L. Y.,
and Patterson, C.
(1999)
Mol. Cell. Biol.
19,
4535-4545 |
33. | Gatto, G. J., Jr., Geisbrecht, B. V., Gould, S. J., and Berg, J. M. (2000) Nat. Struct. Biol. 7, 1091-1095[CrossRef][Medline] [Order article via Infotrieve] |
34. | Kumar, A., Roach, C., Hirsh, I. S., Turley, S., deWalque, S., Michels, P. A., and Hol, W. G. (2001) J. Mol. Biol. 307, 271-282[CrossRef][Medline] [Order article via Infotrieve] |
35. | Callebaut, I., Renoir, J. M., Lebeau, M. C., Massol, N., Burny, A., Baulieu, E. E., and Mornon, J. P. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6270-6274[Abstract] |
36. |
Silverstein, A. M.,
Galigniana, M. D.,
Chen, M. S.,
Owens-Grillo, J. K.,
Chinkers, M.,
and Pratt, W. B.
(1997)
J. Biol. Chem.
272,
16224-16230 |
37. |
Davies, T. H.,
Ning, Y. M.,
and Sanchez, E. R.
(2002)
J. Biol. Chem.
277,
4597-4600 |