©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Taxol Binding Site on the Microtubule
2-(m-AZIDOBENZOYL)TAXOL PHOTOLABELS A PEPTIDE (AMINO ACIDS 217-231) of beta-TUBULIN (*)

(Received for publication, May 12, 1995; and in revised form, June 28, 1995)

Srinivasa Rao (1) George A. Orr (1) Ashok G. Chaudhary (2) David G. I. Kingston (2) Susan Band Horwitz (1)(§)

From the  (1)Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461 and the (2)Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [^3H]3`-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin (Rao, S., Krauss, N. E., Heerding, J. M., Swindell, C. S., Ringel, I., Orr, G. A., and Horwitz, S. B.(1994) J. Biol. Chem. 269, 3132-3134). The interaction of a second photoaffinity analogue of taxol, [^3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels beta-tubulin and the photolabeling is competed by both taxol and unlabeled 2-(m-azidobenzoyl)taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [^3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of beta-tubulin as the major photolabeled domain.


INTRODUCTION

Taxol is an antitumor drug that is used in the treatment of advanced ovarian and breast carcinomas (1, 2) and is demonstrating encouraging activity in a variety of human malignancies(3) . Several important characteristics including its complex chemical structure(4) , its unusual mechanism of action(5) , and its activity in solid tumors, have separated taxol from other conventional antineoplastic drugs. Taxol is an antimitotic drug that also enhances the polymerization of tubulin. Microtubules that are formed in the presence of taxol are resistant to depolymerization by cold temperature, dilution, and Ca. In cells, incubation with taxol results in the formation of stable bundles of microtubules(6) . In vitro, in the presence of taxol, tubulin will polymerize in the absence of GTP, which under normal conditions is an absolute requirement for microtubule polymerization(7, 8) . In contrast to other antimitotic agents such as colchicine and vinblastine, which bind to the tubulin dimer, taxol has a binding site on the microtubule polymer. Taxol binds to microtubules specifically and reversibly with a stoichiometry, relative to the tubulin heterodimer, approaching one(9, 10) .

Our understanding of the binding site for taxol on the microtubule remains ill-defined. In the absence of a crystal structure for the tubulin heterodimer, we have selected photoaffinity labeling as a method to address the nature of the interaction between taxol and its target protein. Earlier studies with taxol demonstrated that the direct photolabeling of tubulin resulted in the preferential labeling of beta-tubulin(11) . However, the low extent of photoincorporation of drug precluded the use of [^3H]taxol in mapping the drug binding sites. To overcome this problem, we have made use of arylazide-containing taxol analogues and have shown previously that [^3H]3`-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-tubulin(12) . In the present study, we have used [^3H]2(m-azidobenzoyl)taxol (2-debenzoyl-2-(4`-[^3H]3`-azidobenzoyl)taxol), a C-2 modified photoaffinity analogue of taxol (Fig. 1). In contrast to 3`-(p-azidobenzamido)taxol in which the arylazide was incorporated into the side chain, this molecule contains the photoreactive group attached to the B ring of the taxoid nucleus. Our results concluded that 2-(m-azidobenzoyl)taxol photolabels a peptide consisting of amino acid residues 217-231 of beta-tubulin.


Figure 1: Molecular structures of taxol, [^3H]2-(m-azidobenzoyl)taxol, and [^3H]3`-(p-azidobenzamido)taxol.




MATERIALS AND METHODS

Taxol and baccatin were obtained from the National Cancer Institute, dissolved in dimethyl sulfoxide, and stored at -20 °C as was 2-(m-azidobenzoyl)taxol. The latter was prepared as described previously(13) . [^3H]2-(m-Azidobenzoyl)taxol was prepared by reductive dehalogenation (14) of a protected 2-(4`-bromo-3`-nitrobenzoyl)taxol; full details of this synthesis will be published elsewhere. Calf brain microtubule protein (MTP) (^1)was purified by two cycles of temperature-dependent assembly-disassembly(15) . The concentration of tubulin in MTP was based on a tubulin content of 85%. Acrylamide/bis, a premixed solution (30:0.8), was purchased from National Diagnostics Inc.; Tricine from Boehringer Mannheim; formic acid, CNBr, SDS, and other biochemicals from Sigma; HiTrap desalting columns from Pharmacia Biotech Inc.; guanidine HCl and Extracti-Gel D from Pierce; and EN^3HANCE from DuPont NEN.

Ultraviolet-induced Cross-linking, SDS-PAGE Analysis, and Fluorography

Photoaffinity labeling studies were done primarily as described with [^3H]3`-(p-azidobenzamido)taxol(12) . [^3H]2-(m-Azidobenzoyl)taxol (10 µM, 0.118 Ci/mmol) was added to MTP (8.5 µM tubulin) in assembly buffer (0.1 M MES, 1 mM EGTA, 0.5 mM MgCl(2), and 3 M glycerol, pH 6.6), incubated at 37 °C for 30 min, and irradiated at 254 nm at 4 °C with a Mineralight lamp (model R52G, UVP Inc., San Gabriel, CA) (0.9 A) at a distance of 7 cm. In addition, the photolabeled samples were analyzed by either analytical (1.5 mm) or preparative (3 mm) SDS-PAGE (9%)(16) . For fluorography, the gels were stained with Coomassie R-250, destained, treated with EN^3HANCE, and exposed to Kodak X-Omat AR film. For isolation of beta-tubulin, the gels were immersed in ice-cold 20 mM KCl for 5 min to visualize the alpha- and beta-tubulin subunits. The beta-tubulin band was excised, washed 6 to 8 times with H(2)O, and electroeluted for 16 h with a Bio-Rad electroeluter (Model 422) at 10 mA per electroelution tube. The recovery of the protein was between 60 and 80%. SDS was removed from electroeluted beta-tubulin using Extracti-Gel D, and the eluate was treated with trichloroacetic acid (final concentration 12%, w/v) to precipitate beta-tubulin and remove SDS. The precipitate was washed twice with ice-cold acetone to remove residual trichloroacetic acid, dried under nitrogen, and dissolved in 100 mM Tris (pH 9.5) containing 5.5 M guanidine HCl. The samples were reduced and carboxymethylated(17) , dialyzed against H(2)O, and lyophilized.

Formic Acid Digestion

Photolabeled carboxymethylated beta-tubulin (10-15 nmol) was dissolved in 400 µl of 75% formic acid and incubated at 37 °C. After 72 h, formic acid was removed by evaporation in a Speed Vac, washed twice with H(2)O, and dried. The formic acid digestion products were separated by SDS-PAGE (17.5%) with 0.1 M Tris, 0.1 M Tricine, and 0.1% SDS as the cathode buffer (18) and visualized by fluorography.

CNBr Digestion

Photolabeled carboxymethylated beta-tubulin (25-30 nmol) was dissolved in 400 µl of 70% formic acid containing CNBr (20 mg) and incubated at 37 °C. After 48 h, the formic acid was evaporated in a Speed Vac, and the sample was washed twice with H(2)O and dried. The material was analyzed by SDS-PAGE (15%) and fluorography. For mapping studies, CNBr-digested beta-tubulin was resuspended in 200 µl of 6 M guanidine HCl containing 2 mM dithiothreitol and chromatographed on a prepacked HiTrap desalting column in the presence of 6 M guanidine HCl. The major radioactive fractions (0.5 ml) were pooled and subjected to reverse phase HPLC on an HP1090 liquid chromatograph using an Aquapore RP-300 C-8 column (2.1 220 mm). The peptides were eluted using a linear acetonitrile, 0.1% trifluoroacetic acid gradient (0-55% over 55 min). The flow rate was 200 µl/min, and 1-min fractions were collected. The recovery of the applied radioactivity was approximately 55%.

Trypsin Digestion

The radioactive fractions isolated from C-8 reverse phase columns were dried, dissolved in 50 mM NH(4)HCO(3), 1 mM CaCl(2), and 1 M urea, and treated with trypsin for 48 h at 25 °C. The total reaction volume was 400 µl, and trypsin was added to the sample in 3 separate additions (at 0, 16, and 25 h), to a final ratio of 1:40 (w/w). 6 M guanidine HCl containing 2 mM dithiothreitol was added to the tryptic digest prior to C-8 reverse phase chromatography. Tryptic peptides were eluted from the column with a linear acetonitrile, 0.1% trifluoroacetic acid gradient (20-40% over 40 min). Individual peaks were collected, and the radioactivity was determined. The radioactive peaks were rechromatographed using an extended acetonitrile gradient (20 to 30% over 40 min). The purified peptide fractions were sequenced on an Applied Biosystems 477A sequenator at the Laboratory of Macromolecular Analysis at the Albert Einstein College of Medicine.


RESULTS AND DISCUSSION

2-(m-Azidobenzoyl)taxol (Fig. 1), a taxol analogue that assembles tubulin in vitro into stable microtubules in the absence of GTP(19) , has been used to photolabel microtubule protein (MTP). Irradiation at 254 nm of 2 cycles of purified MTP with [^3H]2-(m-azidobenzoyl)taxol resulted in the specific photolabeling of beta-tubulin. Neither alpha-tubulin nor microtubule-associated proteins were labeled under our experimental conditions (Fig. 2). A time course demonstrated that the efficiency of photoincorporation of [^3H]2-(m-azidobenzoyl)taxol into beta-tubulin reached a maximum of 0.1-0.15 mol of analogue/mol of tubulin dimer after 10 min of irradiation. Increasing the time of irradiation did not improve the efficiency of photolabeling (data not shown). The extent of photoincorporation was calculated, based on one drug binding site per dimer, by a filter binding assay after precipitation with cold acetone. The addition of a 50-fold molar excess of taxol or unlabeled 2-(m-azidobenzoyl)taxol to the incubation mixture prior to photolabeling, specifically competed with [^3H]2-(m-azidobenzoyl)taxol photoincorporation into beta-tubulin (Fig. 2). The data indicate that taxol was not as good a competitor of photoincorporation as the unlabeled photoaffinity analogue. This observation correlates with the fact that 2-(m-azidobenzoyl)taxol is more potent than taxol in enhancing tubulin polymerization(13, 19) . Due to the extreme hydrophobicity of taxol, it is not possible to use more than a 50-fold molar excess of taxol in this experiment. Under the same experimental conditions, baccatin which does not have the same biological activity as taxol(20) , did not influence the photolabeling reaction. This suggests that taxol and its C-2 modified analogue are binding at the same, or overlapping, sites.


Figure 2: [^3H]2-(m-Azidobenzoyl)taxol specifically photolabels beta-tubulin. MTP (2.5 µM tubulin) was incubated for 10 min at 37 °C with either no additions or with 50 µM taxol, 2-(m-azidobenzoyl)taxol, or baccatin. To each sample, [^3H]2-(m-azidobenzoyl)taxol (1 µM) was added and incubated for an additional 30 min. After the incubation, the samples were irradiated for 10 min and analyzed by SDS-PAGE and fluorography as described under ``Materials and Methods.'' A, Coomassie-stained gel; B, fluorograph of A. Lane 1, no UV irradiation; lane 2, no additions; lane 3, 50 µM taxol; lane 4, 50 µM unlabeled 2-(m-azidobenzoyl)taxol; and lane 5, 50 µM baccatin. The gel was exposed for 17 days for fluorography.



To locate the photolabeled domain within beta-tubulin, [^3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin was electroeluted from gels, reduced, carboxymethylated, and subjected to either formic acid or CNBr cleavage. Formic acid is known to preferentially cleave Asp-Pro bonds(21) . Since beta-tubulin contains two such linkages at positions 31-32 and 304-305(22, 23) , complete formic acid digestion of beta-tubulin resulted in three distinct peptide fragments consisting of amino acids 1-31 (A(1), mass = 3.5 kDa); 32-304 (A(2), mass = 31 kDa), and 305-445 (A(3), mass = 16 kDa)(12) . The peptides resulting from formic acid digestion of [^3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin were separated by SDS-PAGE. The fluorograph of the gel demonstrated that the radiolabel was associated with the A(2) fragment. A(1) and A(3) fragments were not labeled under our experimental conditions. The CNBr digestion of photolabeled beta-tubulin produced a single radiolabeled peptide of approximately 6.5 kDa (Fig. 3B).


Figure 3: Chemical cleavage of [^3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin. The photolabeled beta-tubulin, after reductive alkylation, was treated with either formic acid (A) or CNBr (B) as described under ``Materials and Methods.'' Digests were subjected to SDS-PAGE analysis and fluorography. Lanes 1, undigested [^3H]2(m-azidobenzoyl)taxol-photolabeled beta-tubulin; lanes 2, [^3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin digested with formic acid for 72 h (A) or with CNBr for 48 h (B). In each lane, approximately 20,000 cpm (A) or 33,000 cpm (B) were loaded. The film was exposed for 12 days (A) or 16 days (B) for fluorography.



The following strategy was used to identify the domain in beta-tubulin into which the 2-m-azidobenzoyl group was photoincorporated. [^3H]2-(m-Azidobenzoyl)taxol-photolabeled beta-tubulin was isolated as described above, digested with CNBr, and fractionated on a HiTrap desalting column to remove the lower molecular weight CNBr fragments. The major radioactive peak, which eluted at the void volume of the column, was pooled and the peptides were resolved by reverse phase HPLC on a C-8 column (Fig. 4). A major peak of radioactivity was eluted between 54 and 60 min. The peak was pooled, dried, resuspended in NH(4)HCO(3) buffer, and subjected to trypsin digestion. The resulting tryptic peptides were separated by reverse phase HPLC (Fig. 5). Radioactivity was associated with two closely eluting peaks identified as A and B in Fig. 5. Each peak was rechromatographed on a C-8 reverse phase column, with an extended acetonitrile gradient (Fig. 6, A and B). The resulting peptides were subjected to N-terminal amino acid sequencing. The following sequence was identified: L-T/A-T-P-T-Y-G-D-L-N-H-L-V-S-A. This sequence is identical with the amino acid positions 217-231 of vertebrate beta-tubulins(24) . In addition to this major peptide, the peak seen in panel A also contained a minor sequence corresponding to amino acid positions 337-350. This contaminating peptide was located within the A(3) fragment of formic acid-digested beta-tubulin, which was never labeled under our experimental conditions (see Fig. 3A). The initial yields of the Edman degradation reaction for each radiolabeled peptide sequenced were in close agreement to the concentrations determined from the specific activity of the incorporated taxol analogue. Therefore, the amino acid residues 217-231 of beta-tubulin were the only domains involved in 2-(m-azidobenzoyl)taxol photolabeling. The loss of tritium during the sequencing procedure did not allow us to identify the modified residue(s). This difficulty has been reported in other photoaffinity labeling studies(25) .


Figure 4: Reverse phase HPLC purification of CNBr peptides. The photolabeled beta-tubulin was subjected to reductive alkylation and was treated with CNBr for 48 h. The peptide digest was passed through a HiTrap desalting column prior to reverse phase HPLC analysis. The peptides were eluted from the C-8 column with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (0 to 55% over 55 min). --, absorbance at 214 nm; --, radioactivity profile.




Figure 5: Reverse phase HPLC purification of tryptic peptides. The major [^3H]2-(m-azidobenzoyl)taxol-photolabeled peptides obtained after CNBr cleavage and reverse phase HPLC (Fig. 4) were pooled, digested with trypsin, and analyzed by reverse phase HPLC. The peptides were resolved with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (20 to 40% over 40 min). Peaks were collected by hand, and an aliquot of each was assayed for radioactivity. The inset is an enlargement of the area that contained the majority of the radioactivity and is identified with an asterisk (*).




Figure 6: Rechromatography of the [^3H]2-(m-azidobenzoyl)taxol-photolabeled tryptic peptides. After fractionation (Fig. 5) of the major radioactive tryptic peptides (A and B), they were repurified by subjecting them individually to reverse phase HPLC on a C-8 column and eluting with a linear gradient of acetonitrile, 0.1% trifluoroacetic acid (20 to 30% over 40 min). --, absorbance at 214 nm; - - -, radioactivity profile.



The two domains of beta-tubulin so far identified as forming molecular contacts with taxol consist of amino acids 1-31 (12) and 217-231 (this study). These domains interact with the 3`-benzamido and the 2-benzoyl groups of the taxol analogues, respectively. If the taxol binding site is located within a single alpha,beta heterodimer, these domains, although far apart in the primary sequence, must be located close enough in the microtubule to interact with the bound taxol. It is interesting to note that in the absence of GTP, the bifunctional reagent, N,N`-ethylenebis(iodoacetamide), that contains a 9-Å spacer arm, cross-links the cysteine at position 12 with one of the cysteines in either position 201 or 211(26) . Alternatively, the taxol binding site could be formed within the contact regions between adjacent alpha,beta heterodimers in the microtubule. Since the crystal structure for the tubulin heterodimer is not available, it is not possible to distinguish between these two possibilities. Whatever the nature of the taxol binding site, it would appear that the high affinity site is found only in oligomeric tubulin (9, 27) . The minimal oligomeric species capable of forming the high affinity binding site is not known.

These two taxol contact domains identified in our studies are highly conserved during evolution (24) and have been shown also to be a part of the colchicine binding site(28) , highlighting the functional importance of these domains in tubulin assembly-disassembly reactions. Although colchicine and taxol have, in some respects, opposite effects in the tubulin/microtubule system, both are antimitotic agents, and, at low concentrations, both affect microtubule dynamics(29, 30) . The binding sites for these two antimitotic agents are present in different forms of tubulin. Colchicine interacts with the heterodimer(31) , whereas taxol binds to the microtubule and to small tubulin oligomers (9, 27) .

Structure-activity studies indicate that other positions in taxol can tolerate modifications without loss of microtubule polymerizing activity(20, 32) . Photoreactive groups at these positions will extend our observations on the nature of the taxol binding site in the microtubule.


FOOTNOTES

*
This research was supported in part by United States Public Health Service Grants CA39821 and Cancer Core Support Grants CA13330 (to S. B. H.), CA56677 (to G. A. O.), and CA48974 (to D. G. I. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, NY 10461. Tel.: 718-430-2163; Fax: 718-829-8705.

(^1)
The abbreviations used are: MTP, microtubule protein; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


ACKNOWLEDGEMENTS

We thank Dr. Ruth Angeletti of the Laboratory of Macromolecular Analysis and Dr. Charles Swindell, Bryn Mawr College, for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.