Interaction of Radicicol with Members of the Heat Shock Protein 90 Family of Molecular Chaperones
Theodor W. Schulte,
Shiro Akinaga,
T. Murakata,
Tsutomu Agatsuma,
Seiji Sugimoto,
Hirofumi Nakano,
Yong S. Lee,
Birgitte B. Simen,
Yair Argon,
Sara Felts,
David O. Toft,
Leonard M. Neckers and
Sreenath V. Sharma
Medicine Branch (T.W.S., L.M.N.) National Cancer Institute
National Institutes of Health Rockville, Maryland 20850
Pharmaceutical Research Institute Kyowa Hakko Kogyo Co.
Ltd. (S.A., T.M., T.A.) Sunto-gun, Shizuoka, Japan 411
Tokyo Research Laboratories (S.S., H.N.) Kyowa Hakko Kogyo
Co. Ltd. Machida-shi, Tokyo 194, Japan Laboratory of
Ocular Therapeutics (Y.S.L.) National Eye Institute Bethesda,
Maryland 20892
Department of Pathology (B.B.S., Y.A.) The
University of Chicago Chicago, Illinois 60637
Department
of Biochemistry and Molecular Biology (S.F., D.O.T.) Mayo Graduate
School Rochester, Minnesota 55905
Department of
Microbiology and Immunology (S.V.S.) University of Tennessee
Memphis, Tennessee 38163
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ABSTRACT
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The Hsp90 family of proteins in
mammalian cells consists of Hsp90
and ß, Grp94, and Trap-1
(Hsp75). Radicicol, an antifungal antibiotic that inhibits various
signal transduction proteins such as v-src, ras, Raf-1, and mos, was
found to bind to Hsp90, thus making it the prototype of a second class
of Hsp90 inhibitors, distinct from the chemically unrelated
benzoquinone ansamycins. We have used two novel methods to immobilize
radicicol, allowing for detailed analyses of drug-protein interactions.
Using these two approaches, we have studied binding of the drug to
N-terminal Hsp90 point mutants expressed by in vitro
translation. The results point to important drug contacts with amino
acids inside the N-terminal ATP/ADP-binding pocket region and show
subtle differences when compared with geldanamycin binding. Radicicol
binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that
resides in the endoplasmic reticulum. In contrast to Hsp90, binding of
radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain
as well as the adjacent negatively charged region. Radicicol also
specifically binds to yeast Hsp90, Escherichia coli HtpG,
and a newly described tumor necrosis factor receptor-interacting
protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90.
Thus, the radicicol-binding site appears to be specific to and is
conserved in all members of the Hsp90 family of molecular chaperones
from bacteria to mammals, but is not present in other molecular
chaperones with nucleotide-binding domains.
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INTRODUCTION
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The heat shock protein 90 (Hsp90) family of proteins in mammalian
cells consists of Hsp90
and ß, Grp94, and Trap-1 (Hsp75) (reviewed
in Ref. 1). The two Hsp90 isoforms exist in the cytosol and the nucleus
and associate with a number of signaling proteins. These proteins
include ligand-dependent transcription factors such as steroid
receptors (2, 3, 4) and aryl hydrocarbon receptors (5, 6),
ligand-independent transcription factors such as myoD (7), mutated p53
(8), and hypoxia-inducible factor 1
(9), tyrosine kinases such as
v-src and (10, 11), src family kinases (12, 13), and serine/threonine
kinases such as Raf-1 (14, 15) and Cdk4 (16). Association of these
signaling proteins with Hsp90 is essential for their stability, correct
intracellular location, and biological activity. Geldanamycin and
herbimycin A, both benzoquinone ansamycin antibiotics, bind
specifically to Hsp90 at its amino-terminal nucleotide-binding site
(17, 18, 19), thereby disrupting its association with a number of the
signaling proteins listed above. Upon dissociation from Hsp90, these
signaling proteins become unstable and are rapidly degraded (20, 21).
Grp94 is an Hsp90 homolog that occurs in the endoplasmic reticulum (ER)
and is involved in protein processing in this compartment (22).
Benzoquinone ansamycins cause a robust ER stress response (23),
probably due to accumulation in the ER of multiple incompletely
glycosylated proteins. Trap-1 (Hsp75) has only recently been described
and displays more homology with the bacterial Hsp90 homolog HtpG than
with either Hsp90 or Grp94 (24, 25). Its function is still under
investigation, but its binding to the tumor necrosis factor (TNF)
receptor (TNFR1) and retinoblastoma protein (Rb) has been reported (24, 25).
Radicicol, a macrocyclic antifungal antibiotic originally isolated from
the fungus Monosporium bonorden (26) was shown to be a
potent tranquilizer of low toxicity (27) and inhibited in
vivo angiogenesis (28). In addition, radicicol has been shown to
suppress cellular transformation by a variety of oncogenes such as
src, ras, and mos (29, 30, 31, 32). In the
case of src-transformed cells, treatment with radicicol was
accompanied by enhanced gelsolin expression (33). In addition, in cells
treated with radicicol, Raf-1, p185erbB2, and mutated p53
proteins became destabilized and subject to proteasome-mediated
proteolysis (32, 34). One of the major intracellular targets of
radicicol is the molecular chaperone, Hsp90. This was demonstrated in
two different ways. First, radicicol, which is structurally dissimilar
to benzoquinone ansamycins, efficiently competed with solid-phase
geldanamycin for binding to Hsp90 (34). Second, biotinylated radicicol
was able to identify Hsp90 in a pseudo-Western blot format (35).
However, neither of these studies directly demonstrated radicicol
binding to Hsp90 in solution, nor did they characterize the
radicicol-binding site. Most recently, cocrystallization of radicicol
with yeast Hsp90 has been reported (36).
To better understand the mechanism of interaction of radicicol with
Hsp90 and its family members, we analyzed the binding of both
biotinylated radicicol bound to streptavidin Sepharose and radicicol
linked to Sepharose beads (Rd-Sepharose) to a series of Hsp90 point and
deletion mutants previously characterized for their ability to bind to
immobilized geldanamycin. Additionally, to determine whether the drug
can also recognize other members of the Hsp90 family, we characterized
the ability of immobilized radicicol to bind wild-type Grp94 and
several Grp94 deletion mutants. Finally, we tested the ability of
immobilized radicicol to bind to Trap-1, the bacterial Hsp90 homolog
HtpG, and yeast Hsp90. All Hsp90 family members display binding of
varying affinities to immobilized radicicol.
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RESULTS
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Both Biotinylated Radicicol and Rd-Sepharose Bind Specifically to
Hsp90 and Grp94 in Solution
Radicicol was derivatized at the C9 position and coupled via an
oxime linkage to either biotin or epoxy amino hexyl
(EAH)-Sepharose. A 12-carbon spacer arm was used to couple
radicicol to biotin, and an 8-carbon spacer arm was used to couple
radicicol to Sepharose. Schematic representations of four biotinylated
radicicol molecules coupled to a streptavidin molecule and of radicicol
covalently coupled to Sepharose are depicted in Fig. 1
. Computer modeling of derivatized
radicicol bound to Hsp90, based on the published coordinates of human
Hsp90 (18), a CHARMM (chemistry at Harvard macromolecular
mechanics)-optimized radicicol derivative (37), and the recently
published crystallographic analysis of radicicol bound to an
amino-terminal yeast Hsp90 fragment (36), predicts a comparable Hsp90
binding mode between native radicicol and its derivative coupled to a
carbon spacer at position C9, since the spacer can be oriented outside
the pocket without disturbing the hydrogen bonding and van der Waals
interactions between radicicol and Hsp90 described by Roe et
al. (36) (data not shown).

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Figure 1. Immobilization of Radicicol Using Biotinylated
Radicicol and Streptavidin Beads (left panel) or
Derivatized Radicicol Covalently Coupled to Sepharose Beads
(right panel)
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Biotinylated radicicol bound to streptavidin Sepharose was able to
affinity isolate both Hsp90 and Grp94 from a cell lysate, and the
binding was competed by preincubation of the lysate with excess
unlabeled radicicol (Fig. 2
) or
geldanamycin (data not shown). In panel A, Hsp90, detected by Western
blot, was competed by preincubating the cell lysate with excess soluble
radicicol. In panel B, both Hsp90 and Grp94 were detectable by
sequential immunoblotting using the same membrane. The amount of Hsp90
and Grp94 detected was directly proportional to the amount of
biotinylated radicicol used. Similar results were obtained when
affinity precipitations were performed from metabolically labeled cell
lysates or if radicicol-associated proteins were analyzed by silver
staining (data not shown).

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Figure 2. Affinity Purification of Hsp90 and Grp94 by
Biotinylated Radicicol Immobilized on Streptavidin Beads
Cell lysates were extracted with biotinylated radicicol bound to
streptavidin beads and detected by immunoblotting. Specificity of Hsp90
purification is demonstrated by absence of binding of the protein to
streptavidin beads alone and competition of binding by soluble
radicicol (A). Also, decreasing amounts of biotinylated radicicol were
used with a constant amount of streptavidin beads. Affinity-purified
Hsp90 and Grp94 were detected by sequential immunoblotting of the same
membrane (B).
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Rd-Sepharose beads were also able to affinity isolate endogenous Hsp90
and Grp94, depicted by silver stain and/or immunoblot, from a human
tumor cell lysate (Fig. 3
). Since
proteins with a long half-life are overrepresented in the assay just
described, we also immunopurified proteins from tumor cells
metabolically labeled with [35S]methionine for 2 h,
which resulted in a strong band of 90 kDa (Hsp90) and a weak band of 97
kDa (Grp94). In each case, chaperone binding to immobilized radicicol
was efficiently competed by preincubation with excess soluble
geldanamycin (or radicicol, data not shown). Hsp90 binding to
biotinylated radicicol was also competed by excess ATP and ADP (Fig. 4
), consistent with the ability of these
nucleotides to block chaperone binding to immobilized geldanamycin
(19).

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Figure 3. Affinity Purification of Proteins on Sepharose
Covalently Bound to Radicicol
Cells metabolically labeled with [35S]methionine, and
unlabeled cells, were extracted with radicicol beads, and purified
proteins were detected by silver stain, autoradiography, or Western
blotting with Hsp90 and Grp94 antibodies. Competition with soluble
geldanamycin was used to show specificity of binding.
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Figure 4. Hsp90 Binding to Biotinylated Radicicol Is Competed
by ATP and ADP
Proteins were immunopurified from cell lysates using biotinylated
radicicol and streptavidin beads and analyzed by immunoblotting with
Hsp90 antibody. Beads without biotinylated radicicol served as a
negative control (first lane). ATP and ADP in increasing concentrations
were used to compete radicicol binding to Hsp90.
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Radicicol Binds to an Amino-Terminal Hsp90 Fragment Containing the
Geldanamycin/Nucleotide-Binding Domain, and Several Point Mutations
within this Domain Abrogate Radicicol Binding
We previously mapped the geldanamycin-binding site of Hsp90 using
immobilized geldanamycin and demonstrated that several point mutations
within the nucleotide-binding domain markedly reduced geldanamycin
binding (19). We now performed a similar analysis using immobilized
radicicol. A deletion mutant expressing the first 221 amino acids of
Hsp90 (
C507) that contains the nucleotide/geldanamycin-binding
domain bound to both biotinylated radicicol and to Rd-Sepharose, and
binding was competed by preincubation with excess soluble radicicol
(Fig. 5
, right panel).
Wild-type Hsp90 as well as the point mutants, R181Q and K190A, bind
well to immobilized geldanamycin and also were affinity purified by
immobilized radicicol (Fig. 5
and Ref. 19). Several point mutants that
poorly bind to immobilized geldanamycin also did not bind to
Rd-Sepharose or biotinylated radicicol. These include D92A, G94D, 3G3V
(a triple-point mutant at G131, G134, and G136) and G182D mutations.
G113D and G131D bound poorly to immobilized radicicol, although both
bind with moderate affinity to immobilized geldanamycin (Fig. 5
and
Ref. 19). In contrast, K111A bound strongly to Rd-Sepharose and
biotinylated radicicol, although its binding to immobilized
geldanamycin is somewhat diminished (Fig. 5
and Ref. 19). An additional
point mutant, E46A, demonstrated affinity for both immobilized
radicicol and geldanamycin equivalent to that of wild-type Hsp90 (Fig. 5
).

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Figure 5. Several Point Mutations in the N-terminal Domain of
Hsp90 Abrogate Binding to Immobilized Radicicol
In vitro translated wild-type Hsp90 and a series of
N-terminal point mutants were tested for binding to immobilized
biotinylated radicicol and radicicol beads. Specificity of binding was
tested by competition with free radicicol. A deletion mutant containing
only the N-terminal 221 amino acids of Hsp90 (C507) was also examined.
Binding of the same in vitro translates to immobilized
geldanamycin is shown for comparison.
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Characterization of Radicicol Binding to Grp94
Grp94 contains an amino-terminal domain very similar to the
nucleotide- binding domain of Hsp90. This domain includes four regions
that characterize an unusual ATP-binding motif shared by the
Topoisomerase II, MutL, and Hsp90 families of proteins (38, 39, 40).
Because both biotinylated radicicol and Rd-Sepharose could affinity
isolate Grp94 and Hsp90 from a cell lysate, we wished to further
investigate the binding characteristics of Grp94 to immobilized
radicicol. First, we examined the ability of soluble radicicol to
compete for the binding of in vitro translated wild-type
Grp94 or Hsp90 to Rd-Sepharose (Fig. 6
, A
and B). Surprisingly, radicicol displayed a 5- to 7-fold weaker
apparent affinity for Grp94 than for Hsp90. A concentration of 50
nM radicicol was required to obtain 50% inhibition of the
binding of Grp94 to immobilized radicicol compared with a concentration
of 10 nM in the case of Hsp90.

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Figure 6. Immobilized Radicicol Has a Higher Apparent
Affinity for Hsp90 than for Grp94
Radicicol beads were used to affinity purify in vitro
translated Hsp90 and Grp94 from reticulocyte lysate. Binding of the
chaperone proteins to the resin was competed by increasing
concentrations of soluble radicicol. Purified proteins were separated
by SDS-PAGE and visualized by autoradiography (A) before densitometry
(B). The figure represents one of two similar experiments.
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Because the affinity of Grp94 for radicicol was weaker than that of
Hsp90, we further characterized the binding of Rd-Sepharose to Grp94 by
examining several Grp94 deletion mutants expressed by in
vitro translation. Figure 7
shows a
schematic of the deletion mutants tested and the autoradiograph after
SDS-PAGE. Rd-Sepharose was able to efficiently isolate wild-type Grp94.
The
H deletion mutant (lacking amino acids 112171) failed to bind
to Rd-Sepharose. These data are not unexpected since this mutant lacks
a significant portion, although not all, of the region homologous to
the Hsp90 nucleotide-binding site (see Fig. 7
). Surprisingly, the
A
deletion mutant (which lacks amino acids 259549) and the
P mutant
(lacking amino acids 281344), which both lack the first negatively
charged domain of Grp94, did not bind to Rd-Sepharose even though they
contain an intact nucleotide/geldanamycin-binding domain. The
B
mutant (amino acids 452671 missing) bound to Rd-Sepharose as
efficiently as the wild-type protein. The mutant Npart2 (expressing
only the first 255 amino acids) did not bind radicicol in this assay,
although the mutant Npart3 (expressing the first 355 amino acids) did.
The mutant Cpart1 (expressing amino acids 256675), which again lacked
the N-terminal domain but contained the first negatively charged
domain, also failed to bind to radicicol. Thus, in contrast to its
binding to Hsp90, radicicol binding to Grp94 requires the
nucleotide-binding domain as well as the adjacent negatively charged
region of this chaperone.

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Figure 7. Binding of Grp94 to Radicicol Requires the
N-Terminal Nucleotide Binding Motif as well as the Adjacent Negatively
Charged Domain
A series of Grp94 deletion mutants were expressed by in
vitro translation and affinity purified from reticulocyte
lysate. Specificity of binding was tested by competition with free
radicicol. The schematic shows the domains affected by
various deletions (see text for details).
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Characterization of Radicicol Binding to Trap-1, HtpG, and Yeast
Hsp90
The tumor necrosis factor type I-associated protein, Trap-1, has
recently been identified (24). Although its function remains unknown,
Trap-1 bears limited homology to Hsp90, with the amino-terminal
nucleotide-binding domain of Hsp90 being almost completely conserved.
Trap-1 protein was expressed either by in vitro translation
in rabbit reticulocyte lysate, or by
isopropyl-1-thio-ß-D-galactopyranoside induction
in E. coli transfected with the Trap-1 expression plasmid
pET-9a-TRAP-1. Rd-Sepharose was able to affinity precipitate Trap-1
protein from bacterial lysate. The inducibly expressed 75-kDa band
visible by silver stain was confirmed to be Trap-1 by N-terminal amino
acid sequencing. Trap-1 binding to immobilized radicicol was
efficiently competed by excess soluble radicicol, the radicicol
derivatives KF49073 (derivatized at the C9 position for coupling to
Sepharose) and KF58333 (which competes effectively with immobilized
geldanamycin for Hsp90 binding), soluble geldanamycin, and ATP (Fig. 8A
). Rd-Sepharose was also able to
specifically affinity isolate Trap-1 in vitro translated in
rabbit reticulocyte lysate (Fig. 8B
). Interestingly, the apparent
affinity of Trap-1 for immobilized radicicol is weaker than that of
either Hsp90 or Grp94 for these drugs (Fig. 8C
). Thus, approximately
100 nM soluble radicicol and 1 µM soluble
geldanamycin are required to reduce Trap-1 binding to Rd-Sepharose by
50%.

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Figure 8. Trap-1 Binds to Radicicol
A, Trap-1 was expressed in E. coli, and bacterial
lysates were incubated with radicicol beads, and affinity-purified
proteins were detected by silver stain. EAH Sepharose beads without
radicicol were used as a negative control (lane 1). A 68-kDa band was
identified as Trap-1 by N-terminal microsequencing. Radicicol,
geldanamycin, ATP, and two radicicol derivatives were used to
competitively inhibit protein binding. B, In vitro
translated Trap-1 was purified from reticulocyte lysate using radicicol
beads and detected by autoradiography after separation of proteins by
SDS-PAGE. Binding was competed by increasing concentrations of
soluble radicicol and geldanamycin. C, Band intensities were quantified
and plotted against concentrations of competing drugs.
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The high level of similarity among Hsp90 family proteins with respect
to radicicol binding was further demonstrated by the observation that
yeast Hsp90 as well as the bacterial HtpG protein were affinity
purified with biotinylated radicicol (Fig. 9
). Specificity of this interaction was
shown using either streptavidin beads without biotinylated radicicol
(lane 1) or competition with free radicicol (lanes 4 and 5) as negative
controls. In addition, radicicol failed to bind to other molecular
chaperones such as Hsp70, BiP, and calreticulin, each of which possess
a nucleotide-binding domain dissimilar to that of Hsp90 (data not
shown). Finally, neither purified MutL nor Topoisomerase II bound to
immobilized radicicol (data not shown), nor did radicicol affect the
ATPase activity of these proteins (W. Yang and Y. Pommier, personal
communication). This observation underscores the specificity of
radicicol for the nucleotide-binding domain of Hsp90 family
members.

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Figure 9. Binding of Radicicol to Yeast Hsp90 and E.
coli HtpG
Yeast cell lysates (A) and cell lysates from E. coli (B)
were affinity precipitated with biotinylated radicicol, resolved by
7.5% SDS PAGE, and either silver stained (left panels
in A and B) or transferred to a polyvinylidene fluoride membrane
and immunoblotted with appropriate antibodies (right
panels in A and B). Lane 1, Streptavidin Sepharose alone; lane
2, streptavidin Sepharose conjugated with 1 µl of biotinylated
radicicol; lane 3, streptavidin Sepharose conjugated with 0.5
µl of biotinylated radicicol. In lanes 4 and 5, cell lysates
were pretreated with 8 nM or 8 µM of native
radicicol, respectively, before affinity precipitation with
streptavidin Sepharose-biotinylated radicicol complex. Approximate
positions of prestained mol wt markers are shown between the two
panels, and the positions of yeast Hsp90 and E. coli
HtpG proteins are indicated by arrows to the right of the
autoradiograms in A and B, respectively.
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DISCUSSION
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The binding and inhibition of Hsp90 by radicicol have far reaching
biological implications. Hsp90, together with its kinase-targeting
entity cdc37 (16), is important in the functioning of several key
signal transducers and cell cycle regulators. There is biochemical
evidence for physical interaction between Hsp90 and many signaling
molecules, including Raf1 (14, 15), v-src (10, 11), Cdk4 (16), wee1
(41), casein kinase II (CKII) (42), and eIF-2
kinase (43), among
others (for a review see Ref. 44). In addition, genetic evidence points
to interactions between Hsp90/cdc37 and molecules such as torso,
sevenless, mps1, kin28, cdc28, and fus3 (reviewed in Ref. 45).
Therefore, radicicol has the potential to impinge on a large array of
signal transduction networks. Consistent with this hypothesis,
radicicol has been shown to suppress cellular transformation by a
number of oncogenes including v-src, Ras, and Mos (29, 30, 31, 32). The
molecular mechanism was shown to involve inhibition of Hsp90, and
radicicol was thus identified as the prototype of a new class of Hsp90
inhibitors chemically unrelated to the benzoquinone ansamycins (34, 35). Because of the importance of Hsp90 inhibitors as research tools
(46, 47), and because of their potential as antitumor agents (1, 48),
we have studied the interaction between radicicol and the various Hsp90
family members in more detail.
The two earlier studies that demonstrated binding of radicicol to Hsp90
depended on indirect approaches (34, 35). Sharma et al.
separated protein lysates by denaturing SDS-PAGE and incubated the
protein transferred to nitrocellulose membranes with biotinylated
radicicol, which was then detected by streptavidin-coupled horseradish
peroxidase. Even though specificity was shown by appropriate
positive and negative controls, the binding of radicicol to Hsp90 after
denaturing electrophoresis might not completely reflect the situation
in vivo. In the study of Schulte et al., binding
of radicicol to Hsp90 was demonstrated by competition of radicicol with
immobilized geldanamycin. We now use two direct approaches to show
binding of Hsp90 to immobilized radicicol under nondenaturing
conditions. As these methods directly assess radicicol binding to
Hsp90, mutants that either do not bind or bind poorly to geldanamycin
can be adequately tested. Our analyses reveal that immobilized
radicicol specifically immunopurifies Hsp90 and that binding is readily
competed by radicicol, geldanamycin, ATP, and ADP.
The marked difference between the structures of radicicol and
geldanamycin and the results of an earlier study (35) had suggested
possible differences in the Hsp90-binding sites of the two drugs. For
this reason, we compared binding of immobilized radicicol to a number
of Hsp90 point mutations that altered amino acids in the N-terminal
nucleotide-binding domain. Asp92 (93 in human Hsp90) lies at the bottom
of the inner face of the nucleotide-binding pocket and forms a hydrogen
bond network with the carbamate group of geldanamycin and a water
molecule (18, 36, 49). As expected, both D92A and G94D mutations cause
a significant reduction in geldanamycin binding. These mutations also
strongly inhibit binding to radicicol. Glu46 (47 in human Hsp90) and
its homologous Glu residues are important for the ATPase function of
gyrase B and Hsp90. Mutations of this residue eliminate gyrase function
in E. coli (50). In yeast Hsp90, mutation of this residue
leads to a protein that can bind but not hydrolyze ATP and cannot
functionally replace wild-type Hsp90 in vivo (51, 52). In
our model system, both immobilized geldanamycin and radicicol clearly
bind to Hsp90 E46D, suggesting that these inhibitors do not require
interaction with a functional ATPase domain on Hsp90.
Lys111 (112 in human Hsp90) makes hydrogen bonds with one of the
benzoquinone oxygen atoms of geldanamycin at the solvent-exposed
entrance to the binding pocket. The K111A and G113D mutations both
partially inhibit binding to geldanamycin. Interestingly, K111A binds
well to radicicol, which does not have a benzoquinone ring, while the
G113D mutation, which presumably leads to changes in the protein
backbone structure of Hsp90, strongly decreases radicicol binding.
The amino acids Gly131, Gly134, and Gly136 form a GXXGXG motif that is
conserved not only in all Hsp90 family proteins but also in bacterial
DNA gyrase B and mutL proteins. In E. coli Gyrase B, two
glycines of this motif are in direct contact with ATP based on the
crystal structure (53), while mutations of this motif in E.
coli mutL produce a dominant mutator phenotype (54). In Hsp90,
these amino acids lie at the opening face of the pocket opposite to
Lys111 and form hydrogen bonds with the magnesium ion that is required
for stabilization of ATP binding (18). Mutation of Gly131 alone does
not completely abrogate geldanamycin binding to Hsp90, although the
mutation of all three glycine residues does abrogate binding. In the
case of radicicol, G131D itself shows a markedly reduced binding,
suggesting important contacts of this residue with radicicol. Arg181,
Gly182, and Lys190 are highly evolutionarily conserved amino acids in
ß-sheet 7, which forms another part of the binding pocket.
R181Q and K190A bind well to geldanamycin and radicicol while G182D
does not, which might be related to a possible disruption in the
backbone of the protein structure. These data are in general agreement
with the recently published crystal structure of radicicol bound to
yeast Hsp90 (36) and, taken together, they demonstrate surprisingly
subtle differences between radicicol and geldanamycin contacts with
this chaperone, even though these drugs are structurally distinct.
Careful examination of benzoquinone ansamycin activity has revealed
that the ER is another site of drug action. For example, both
geldanamycin and radicicol have been shown to be potent inducers of the
ER stress response, leading to transcriptional up-regulation of ER
chaperones (Ref. 23 and L. Hendershot, personal communication).
For this reason, we wanted to compare radicicol binding to Grp94 with
its binding to Hsp90. Grp94 was specifically bound by immobilized
radicicol, and binding was competed by both soluble radicicol and
geldanamycin. However, radicicol displayed a 5-fold higher apparent
affinity for Hsp90 than for Grp94.
While mapping the Grp94 radicicol-binding site with a series of
deletion mutants, we identified a major difference in drug binding
compared with Hsp90. Not surprisingly, a deletion in the N-terminal
domain that includes the sequence homologous to the nucleotide-binding
pocket of Hsp90 led to abrogation of radicicol binding to Grp94.
Interestingly, a deletion of the adjacent negatively charged domain,
which follows the putative nucleotide-binding pocket after a linking
region, also abrogated radicicol binding even though the
nucleotide-binding domain remained intact. A deletion in the more
C-terminal part of the molecule that included the second negatively
charged domain of Grp94 did not interfere with radicicol binding. The
importance of the amino-terminal charged domain for radicicol binding
is emphasized by the fact that a deletion mutant that only expresses
the N-terminal domain of the molecule containing the putative
nucleotide-binding site does not bind radicicol, while a mutant that
expresses both the N-terminal domain and the first negatively charged
domain does bind to immobilized drug. This represents a clear
difference with Hsp90, where a mutant expressing only the N-terminal
domain containing the ATP/ADP binding pocket was bound by immobilized
radicicol. The apparent requirement of an additional domain for
radicicol binding to Grp94 suggests that novel drug-protein contacts
may be made, although confirmation of this hypothesis awaits the
crystallization of Grp94. If so, it would appear theoretically possible
to design radicicol derivatives with specificity for either Hsp90 or
Grp94.
Trap-1 (Hsp75) is a recently described member of the Hsp90 family
of proteins that resembles the bacterial HtpG protein in both size and
structural organization (24, 25). The function of this protein in
mammalian cells still remains to be determined, but it may be of
importance to tumor cell biology since it binds to the type 1 TNF
receptor (TNFR1) (24) and the retinoblastoma protein (25). Trap-1
shares the N-terminal nucleotide-binding domain of Hsp90 and Grp94 but
lacks the charged region of Hsp90 and differs in the C terminus. Our
experiments with immobilized radicicol demonstrate binding of Trap-1 to
the drug, and binding is competed by excess radicicol, geldanamycin,
and ATP. However, the apparent affinity of radicicol for Trap-1 is
10-fold less than for Hsp90 and half that for Grp94.
The N-terminal nucleotide-binding domain is highly conserved throughout
the Hsp90 family, as emphasized by the fact that radicicol binds to all
members of the family so far examined, from bacteria to man. This
uniquely structured nucleotide binding site differs from other more
common ATP-binding motifs. Intriguingly, although this motif is
conserved in the gyrase B and MutL families, neither radicicol nor
geldanamycin bind to or affect the activity of these proteins.
Hsp90-inhibitory drugs that bind to this domain lock the protein into a
conformation that is similar to its ADP-bound state. In Hsp90,
nucleotide binding seems to function as a molecular switch regulating
two opposing conformational states, which, in turn, have distinct
binding affinities for a series of chaperones and cochaperones critical
to Hsp90 function. However, the function of this domain in other Hsp90
family members remains to be determined.
 |
MATERIALS AND METHODS
|
---|
Antibodies and Reagents
Radicicol and radicicol derivatives were supplied by
the Pharmaceutical Research Institute (Kyowa Hakko Kogyo Co. Ltd.,
Shizuoka, Japan). Biotinylated radicicol, described previously (35),
was supplied by Tokyo Research Laboratories (Kyowa Hakko Kogyo Co.
Ltd., Machida, Japan). Geldanamycin was obtained from the Developmental
Therapeutics Program, National Cancer Institute (Rockville, MD). Drugs
were dissolved in dimethylsulfoxide (DMSO) as 10 or 5 mM
stock solutions. Antibodies used were directed against Hsp90 [either
Ab-1 from Lab Vision (Freemont, CA) or AC88 from Stress Gen (Vancouver,
British Columbia, Canada)] and Grp94 (either Ab-1, from Lab Vision, or
a kind gift from Dr. Linda Hendershot, St. Jude Childrens Hospital,
Memphis TN). Antibodies specific for yeast Hsp90 and E. coli
HtpG were kindly provided by Dr. Susan Lindquist (University of
Chicago, Chicago, IL) and Dr. Michael Ehrmann (University of Konstanz,
Konstanz, Germany), respectively. All other reagents were of
highest available grade.
Plasmid Constructions
pGEM99.2 encoding a full-length wild-type mouse GRP94 cDNA in
pGEM3 was a kind gift from Dr. Michael Green (55). pGEM3-d
A was
constructed by partial digestion of pGEM99.2 with AlwNI and
religation of the 4.8-kb fragment. pGEM3-
B was constructed by
digestion of pGEM99.2 with BclI and religation of the 5.0-kb
fragment. pGEM3-
H was constructed by partial digestion of pGEM99.2
with HpaI and MscI and religation of the 5.4-kb
fragment. Subsequently, a region corresponding to amino acids 1524 of
the full-length protein was amplified by PCR, sequenced, and subcloned
back into pGEM-
H using XbaI and XhoI to
produce pGEM3-
H-ss. pGEM3-
P was constructed by partial digestion
of pGEM99.2 with PstI and religation of the 5.4-kb fragment.
Subsequently, a region corresponding to amino acids 1132 of the
full-length protein was amplified by PCR, sequenced, and subcloned back
into pGEM3-
P using XbaI and HpaI to produce
pGEM3-
P-ss. To construct pGEM-Npart2, a region corresponding to
amino acids -21 to 255 was amplified by PCR and subcloned into
pGEM-T Easy (Promega Corp., Madison, WI). pGEM-Npart3 and
pGEM-Cpart1 were constructed in analogous fashion using PCR products
spanning amino acids -21 to 355 and 256675, respectively. The
antisense primers for the latter three constructs all contained the ER
retention signal KDEL.
Point and deletion mutants of Hsp90 were as described in Ref. 19 .
pBluescript-Trap-1 encoding the full-length wild-type Trap-1 cDNA was
generously provided by D. B. Donner (Indiana University School of
Medicine, Indianapolis, IN) (24).
Cell Culture and Preparation of Lysates
SKBR3 cells were obtained from the American Type Culture Collection (Manassas, VA). The cells were maintained in DMEM
with 10% FCS and 10 mM HEPES. For metabolic labeling,
cells were washed with PBS and kept in methionine-free medium for 30
min before the addition of 100 µCi/ml [35S]methionine
for 2 h. Cells were lysed with TNES buffer (50 mM
Tris-HCl, pH 7.5, 1% NP40, 2 mM EDTA, 100 mM
NaCl) containing 1 mM sodium orthovanadate, 20 µg/ml
aprotinin, 20 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride (PMSF).
RAS-3T3 cells were maintained as described previously (35). For
affinity precipitations using the biotinylated radicicol reagent,
2-day-old subconfluent cells were washed once with ice-cold PBS. Cells
were then resuspended in hypotonic lysis buffer RSBT (10 mM
Tris, pH 7.6, 10 mM NaCl, 1.5 mM
MgCl2, 0.1% Tween, 1 mM sodium orthovanadate,
10 µg/ml leupeptin, 10 µg/ml aprotinin, 5 mM PMSF.
Cells were allowed to swell in RSBT for 10 min, after which they were
homogenized in a Dounce homogenizer (B pestle) with 30 strokes.
Unbroken cells were removed by a slow speed spin (2000 rpm/4 C for 10
min), and the resulting lysates were then clarified by a hard spin
(microfuge, 4 C for 30 min).
Yeast strain GYC86 was grown to saturation, at which time cells were
collected by centrifugation and resuspended in SORB buffer (0.3
M Sorbitol, 100 mM NaCl, 5 mM
MgCl2, 10 mM Tris, pH 7.4, 1 mM
sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 5
mM PMSF). Cells were lysed by vortexing with glass beads
(10 times, 30 sec each), and the unbroken cells were removed by
low-speed centrifugation (6000 rpm for 10 min). The cell lysates were
clarified by a hard spin (microfuge, 4 C for 30 min). Supernatants were
collected and subjected to affinity precipitation analysis with
biotinylated radicicol. E. coli strain DH1 was grown to
saturation, at which time cells were collected by centrifugation and
resuspended in RSBT containing 1 mM dithiothreitol. Cells
were subjected to a cycle of freeze-thaw followed by sonication (10
times, 30 sec each). The unbroken cells were removed by low-speed
centrifugation (6000 rpm for 10 min). The cell lysates were clarified
by a hard spin (microfuge, 4 C for 30 min), and supernatants were
collected and subjected to affinity precipitation analysis with
biotinylated radicicol.
Preparation and Use of Biotinylated Radicicol-Streptavidin
Sepharose Complex
Biotinylated radicicol (KT8529) was resuspended in DMSO to yield
a stock solution of 10 mM. Titrations were performed to
determine the amount of KT8529 that would be sufficient to saturate 25
µl of packed Streptavidin Sepharose beads (Zymed Laboratories, Inc., South San Francisco, CA) (Fig. 2B
, top panel).
Based on the result of this titration, 1 µl of the KT8529 was added
to 50 µl of Streptavidin Sepharose beads (50% solution) and
incubated in the dark at room temperature for 10 min. Unbound KT8529
was removed by washing the beads three times with PBS containing 0.2%
Tween (PBST). The biotinylated radicicol-Streptavidin Sepharose beads
(BR-SS) were used as an affinity matrix to capture radicicol-binding
proteins from cell lysates. Briefly, 1.5 ml of RSBT cell lysates
(equivalent to one subconfluent 100-mm dish of cells) were incubated
with the BR-SS affinity matrix. In some cases where competition was
examined, the cell lysates were preincubated with varying
concentrations of natural radicicol (UCS1006) or geldanamycin for
2 h before incubation with the BR-SS affinity matrix. Proteins
bound to the affinity matrix were collected by brief centrifugation,
and the beads were washed three times with PBST, resuspended in 50 µl
of Laemmlis sample buffer, boiled, and separated by 7.5% SDS-PAGE
(56). Electrophoretically separated proteins were transferred to
polyvinylidene fluoride membranes and probed with
-HSP90
antibody (1:1000 dilution),
-GRP94 antibody (1:2000 dilution),
-yeast HSP90 antibody (1:4000 dilution), or
-HtpG antibody
(1:20,000 dilution) in blocking solution (90 min at room temperature).
Filters were then washed briefly with PBST and incubated with either
horseradish peroxidase-conjugated goat
-rabbit or goat
-mouse
antibodies (1:4000 dilution) in blocking solution (60 min at room
temperature) and visualized by enhanced chemiluminescence (ECL
detection kit, Amersham Pharmacia Biotech, Arlington
Heights, IL) as described (30).
Production and Use of Radicicol and Geldanamycin Affinity
Beads
Purified radicicol derivative KF66658 (25 mg) was dissolved in
tetrahydrofuran and added to 1 ml of EAH-Sepharose beads
(Pharmacia Biotech, Piscataway, NJ). After 5 days
of end-over-end mixing at room temperature, the beads were transferred
into tetrahydrofuran-methanol (1:1), and 20 mg of acetic anhydride were
added. After end-over-end mixing at room temperature for 1 h, 2
vol of 1 M Tris-HCl (pH 7.5) were added for 15 min.
Finally, the resin was washed three times in TNES buffer and blocked in
1% BSA before use. Geldanamycin was derivatized and immobilized as
previously reported (17).
Recombinant proteins were expressed by in vitro
transcription/translation using the TNT rabbit reticulocyte lysate kit
(Promega Corp.) in the presence of 1458 Ci/mmol
translation grade [35S]methionine (ICN Biochemicals, Inc., Aurora, OH) using the appropriate DNA
polymerase and following the manufacturers instructions. Material
from in vitro translation reactions (1224 µl) or
cellular lysates (100 µg of total protein) were incubated with
various concentrations of radicicol or geldanamycin. After mixing at 4
C for 30 min, 40 µl of resin with immobilized radicicol or
geldanamycin were added and incubated for 60 min at 4 C with
end-over-end mixing. Resins were washed three times with TENS buffer
and boiled in Laemmli sample buffer (56). After separation of proteins
by SDS-PAGE, proteins were visualized by silver stain, immunoblotting,
or autoradiography. Western blotting was performed as previously
described (57). We used horseradish peroxidase-conjugated secondary
antibody to rabbit or mouse IgG (Amersham Pharmacia Biotech) in conjunction with Western blot chemiluminescence
reagent (Renaissance, DuPont Merck Pharmaceutical Co.,
Wilmington, DE). Films were scanned into a Macintosh computer
and processed using Adobe Photoshop (Adobe Systems, Inc., San Jose, CA)
and NIH image (NIH, Bethesda, MD) software.
Analysis of Trap-1-Radicicol Binding
Trap-1 was expressed in E. coli BL21(DE3) using the
inducible expression vector pET-9a-Trap-1. Induced bacteria were
suspended in PBS, disrupted by sonication, and diluted in TNES buffer
containing 1 mM sodium orthovanadate and 1 mM
dithiothreitol. After centrifugation, 500 µl of supernatant were
incubated with 10 µl of the solvent DMSO or competitors for 45 min
before adding EAH Sepharose beads or radicicol beads for 45 min. After
two washes with TNES buffer and one wash with TE buffer, pellets were
boiled in Laemmli sample buffer and used for SDS-PAGE. The identity of
an affinity-purified protein of 68 kDa size was determined by
N-terminal amino acid sequencing. Alternatively, Trap-1 was in
vitro translated from pBluescript-Trap-1 in rabbit reticulocyte
lysate and affinity purified using Rd-Sepharose beads as described
above.
 |
ACKNOWLEDGMENTS
|
---|
We gratefully thank D. B. Donner (Indiana University School
of Medicine, Indianapolis, IN) for pBluescript-Trap-1 encoding the
full-length wild-type Trap-1 cDNA, and Tali Gidalevitz (University of
Chicago, Chicago, IL) for expert technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Len Neckers, Ph.D., Theodor W. Schulte, M.D., Medicine Branch, National Cancer Institute, 9610 Medical Center Drive, Suite 300, Rockville, Maryland 20850.
Received for publication March 17, 1999.
Revision received May 20, 1999.
Accepted for publication May 25, 1999.
 |
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