(Received for publication, May 12, 1995; and in revised form, June 28, 1995)
From the
Photoaffinity labeling methods are being used to define the
molecular contacts between taxol and its target protein, tubulin. Our
laboratory has demonstrated previously that
[H]3`-(p-azidobenzamido)taxol
photolabels the N-terminal 31 amino acids of
-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, [
H]2-(m-azidobenzoyl)taxol, with
tubulin has been investigated. This analogue specifically photolabels
-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,
[
H]2-(m-azidobenzoyl)taxol-photolabeled
-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
-tubulin as the major photolabeled domain.
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
-tubulin(11) . However, the low extent of
photoincorporation of drug precluded the use of
[
H]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
[
H]3`-(p-azidobenzamido)taxol
photolabels the N-terminal 31 amino acids of
-tubulin(12) . In the present study, we have used
[
H]2(m-azidobenzoyl)taxol
(2-debenzoyl-2-(4`-[
H]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
-tubulin.
Figure 1:
Molecular structures of taxol,
[H]2-(m-azidobenzoyl)taxol, and
[
H]3`-(p-azidobenzamido)taxol.
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) .
[H]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) (
)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
HANCE from DuPont NEN.
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
[H]2-(m-azidobenzoyl)taxol resulted in
the specific photolabeling of
-tubulin. Neither
-tubulin nor
microtubule-associated proteins were labeled under our experimental
conditions (Fig. 2). A time course demonstrated that the
efficiency of photoincorporation of
[
H]2-(m-azidobenzoyl)taxol into
-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
[
H]2-(m-azidobenzoyl)taxol
photoincorporation into
-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:
[H]2-(m-Azidobenzoyl)taxol
specifically photolabels
-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,
[
H]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 -tubulin,
[
H]2-(m-azidobenzoyl)taxol-photolabeled
-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
-tubulin contains two such linkages at positions 31-32 and
304-305(22, 23) , complete formic acid digestion
of
-tubulin resulted in three distinct peptide fragments
consisting of amino acids 1-31 (A
, mass =
3.5 kDa); 32-304 (A
, mass =
31 kDa),
and 305-445 (A
, mass =
16
kDa)(12) . The peptides resulting from formic acid digestion of
[
H]2-(m-azidobenzoyl)taxol-photolabeled
-tubulin were separated by SDS-PAGE. The fluorograph of the gel
demonstrated that the radiolabel was associated with the A
fragment. A
and A
fragments were not
labeled under our experimental conditions. The CNBr digestion of
photolabeled
-tubulin produced a single radiolabeled peptide of
approximately 6.5 kDa (Fig. 3B).
Figure 3:
Chemical cleavage of
[H]2-(m-azidobenzoyl)taxol-photolabeled
-tubulin. The photolabeled
-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
[
H]2(m-azidobenzoyl)taxol-photolabeled
-tubulin; lanes 2,
[
H]2-(m-azidobenzoyl)taxol-photolabeled
-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 -tubulin into which
the 2-m-azidobenzoyl group was photoincorporated.
[
H]2-(m-Azidobenzoyl)taxol-photolabeled
-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
HCO
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
-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
fragment of formic
acid-digested
-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
-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 -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
[H]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
[H]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 -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
,
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
,
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.