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Molecular Endocrinology 13 (9): 1435-1448
Copyright © 1999 by The Endocrine Society

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Hsp90 family of proteins in mammalian cells consists of Hsp90 {alpha} 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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The heat shock protein 90 (Hsp90) family of proteins in mammalian cells consists of Hsp90{alpha} 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{alpha} (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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go. 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)

 
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. 2Go) 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).

 
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. 3Go). 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. 4Go), 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.

 
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 ({Delta}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. 5Go, 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. 5Go 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. 5Go and Ref. 19). In contrast, K111A bound strongly to Rd-Sepharose and biotinylated radicicol, although its binding to immobilized geldanamycin is somewhat diminished (Fig. 5Go and Ref. 19). An additional point mutant, E46A, demonstrated affinity for both immobilized radicicol and geldanamycin equivalent to that of wild-type Hsp90 (Fig. 5Go).



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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.

 
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. 6Go, 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.

 
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 7Go 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 {Delta}H deletion mutant (lacking amino acids 112–171) 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. 7Go). Surprisingly, the {Delta}A deletion mutant (which lacks amino acids 259–549) and the {Delta}P mutant (lacking amino acids 281–344), 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 {Delta}B mutant (amino acids 452–671 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 256–675), 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).

 
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. 8AGo). Rd-Sepharose was also able to specifically affinity isolate Trap-1 in vitro translated in rabbit reticulocyte lysate (Fig. 8BGo). Interestingly, the apparent affinity of Trap-1 for immobilized radicicol is weaker than that of either Hsp90 or Grp94 for these drugs (Fig. 8CGo). 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.

 
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. 9Go). 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.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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{alpha} 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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 Children’s 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{Delta}A was constructed by partial digestion of pGEM99.2 with AlwNI and religation of the 4.8-kb fragment. pGEM3-{Delta}B was constructed by digestion of pGEM99.2 with BclI and religation of the 5.0-kb fragment. pGEM3-{Delta}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 1–524 of the full-length protein was amplified by PCR, sequenced, and subcloned back into pGEM-{Delta}H using XbaI and XhoI to produce pGEM3-{Delta}H-ss. pGEM3-{Delta}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 1–132 of the full-length protein was amplified by PCR, sequenced, and subcloned back into pGEM3-{Delta}P using XbaI and HpaI to produce pGEM3-{Delta}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 256–675, 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. 2BGo, 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 Laemmli’s sample buffer, boiled, and separated by 7.5% SDS-PAGE (56). Electrophoretically separated proteins were transferred to polyvinylidene fluoride membranes and probed with {alpha}-HSP90 antibody (1:1000 dilution), {alpha}-GRP94 antibody (1:2000 dilution), {alpha}-yeast HSP90 antibody (1:4000 dilution), or {alpha}-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 {alpha}-rabbit or goat {alpha}-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 manufacturer’s instructions. Material from in vitro translation reactions (12–24 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.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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