(Received for publication, November 16, 1995; and in revised form, February 21, 1996)
From the
The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine
(3CTC) is a competitive inhibitor of colchicine binding to tubulin,
binds to tubulin at 37 °C, but not at 0 °C, and covalently
reacts with -tubulin at 37 °C, but not at 0 °C, in a
reaction inhibited by colchicine site drugs. The approximate
intramolecular distance between the oxygen at position C-3 in 3CTC and
the chlorine atom of the 3-chloroacetyl group is 3 Å. Using
decylagarose chromatography, we purified
-tubulin that had reacted
with
3-(chloromethyl-[
C]carbonyl)-3-demethylthiocolchicine
([
C]3CTC). This
-tubulin was digested with
formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase
Lys-C, and the radiolabeled peptide(s) were isolated. The sequences of
these peptides indicated that as much as 90% of the covalent reaction
between the [
C]3CTC and
-tubulin occurred
at cysteine 354. This finding indicates that the C-3 oxygen atom of
colchicinoids is within 3 Å of the sulfur atom of the Cys-354
residue, suggests that the colchicine A ring lies between Cys-354 and
Cys-239, based on the known 9 Å distance between these residues,
and may indicate that the tropolone C ring lies between the peptide
region containing Cys-239 and the amino-terminal
-tubulin
sequence, based on the labeling pattern observed following direct
photoactivation of tubulin-bound colchicine.
Tubulin interacts with a wide variety of small ligands,
including divalent cations, guanine nucleotides, and antimitotic drugs.
Perhaps because of its instability, its tendency to polymerize into
pleomorphic structures, and/or its microheterogeneity, tubulin or
tubulin-ligand complexes have not been crystallized. A polymeric site
for paclitaxel(1) , probably on the
-subunit(2, 3) , has been defined in zinc-induced
sheets of antiparallel protofilaments. Efforts to define ligand binding
sites have generally involved induction of covalent interactions
between tubulin and specific ligands. Usually photoreactive derivatives
of the ligand were used (e.g.(2, 3, 4, 5) ), but
``direct photoaffinity'' labeling, in which a covalent bond
between ligand and tubulin forms under UV irradiation, has also been a
valuable technique(6, 7, 8) . In most cases
the target amino acids in the covalent interaction between protein and
ligand were not identified, with localization of the reactive site
limited to the tubulin subunit or a peptide fragment.
No ligand has
attracted more attention than colchicine(9) , whose interaction
with tubulin permitted the initial isolation of the protein (10) . Photoaffinity (4, 5) and direct
photoaffinity (7, 11) studies have provided evidence
that both the - and
-subunits participate in forming the
binding site, as has genetic
evidence(12, 13, 14) . In only one of these
studies (11) was label localized to specific peptide fragments
of tubulin, and the region of colchicine that reacted with the protein
was uncertain.
We have taken a different approach from concern that use of bulky photoaffinity groups introduces a paradox into the technique. Adequate affinity of such compounds for tubulin implies that the reactive group has been introduced at a position in the ligand of secondary importance for the binding reaction. Thus, covalent reaction(s) may occur at some distance from the binding site itself. While this problem does not arise with the direct photoaffinity method, the precise ligand-peptide reaction has only been established with GTP(6) . We decided to explore the use of small, chemically reactive substituents introduced at defined positions in colchicinoids and allocolchicinoids(15, 16) . Schmitt and Atlas (17) had used an analog derivatized with the bromoacetyl group in the B ring side chain, but they found extensive nonspecific covalent reactions between their analog and tubulin. We used the isothiocyanate and chloroacetyl groups(16) , and introduced them into the side chain, at position C-9 of the C ring in allocolchicinoids or at position C-2 or C-3 of the A ring in thiocolchicine. While the side chain and C ring derivatives retained high affinity for tubulin, extensive nonspecific (i.e. not inhibited by colchicine site agents) covalent reactions occurred with these compounds, and we abandoned them as unsuitable for further study.
The A ring
derivatives, 2CTC ()and 3CTC (structure in Fig. 1),
were much more promising(18) . Both agents were similar to
colchicine in their interactions with tubulin and were competitive
inhibitors of the binding of [
H]colchicine to
tubulin. Stoichiometry of binding of both derivatives was comparable
with that of colchicine, and with both analogs 80% of the covalent
reaction was with
-tubulin. The 3CTC reacted rapidly once bound,
with 60% of the bound 3CTC forming a covalent bond, while the 2CTC
covalent reaction lagged kinetically behind the binding reaction, with
only one-fourth of bound agent reacting with tubulin. The binding and
covalent reactions occurred minimally at 0 °C and were strongly
inhibited by podophyllotoxin. Here we demonstrate that the covalent
reaction of 3CTC with
-tubulin occurs primarily at Cys-354.
Figure 1:
Separation of the - and
-subunits of tubulin on decylagarose following interaction of the
protein with [
C]3CTC. The sample in Solution A
was initially applied to a column equilibrated with Solution A. At the
point indicated by the arrow Solution A was replaced with 4 M guanidine HCl (pH 5.0) as the eluting solution. Sample size:
25 mg of tubulin. Fraction volume: 5 ml. Solid symbols represent protein, determined on 100-µl aliquots. Open
symbols represent radiolabel, determined on 100-µl aliquots.
Polyacrylamide gel electrophoresis was performed on 10 µg of the
pooled material from each peak, with electrophoretic transfer to a PVDF
membrane. The stained membrane is shown in the figure. The structure of
3CTC is also shown in the figure.
For enzyme digestions(23) , 3CTC-peptide complexes were dissolved in 1% SDS for 1 h at 37 °C or by boiling for 5 min. These solutions were diluted 10-fold into the proteinase solutions at an enzyme to substrate ratio of 1:100 and incubated for 24-48 h at 37 °C. With endoproteinase Lys-C the buffer was 0.1 M boric acid-borax (pH 7.6), with endoproteinase Glu-C, 50 mM ammonium acetate (pH 4.0). If digestion was judged to be incomplete, additional enzyme at 1:100 was added to the reaction mixture, which was incubated at 37 °C for 24 h.
Denatured
tubulin subunits can be resolved by hydrophobic chromatography on
decylagarose(20) . Fig. 1summarizes a separation
following reaction with [C]3CTC. In the presence
of 2 M NaCl, 4 M guanidine HCl the column only binds
-tubulin, which is eluted by 4 M guanidine HCl without
NaCl. Fig. 1also shows SDS-polyacrylamide gels of the unbound
(on the left) and bound (on the right) protein. As
before(18) , most of the radiolabel that had reacted covalently
with tubulin was associated with the unbound
-tubulin. In the
preparation shown in Fig. 1, there was over five times as much
radiolabel associated with
-tubulin as with
-tubulin, with
drug/monomer stoichiometries of 0.32 and 0.06, respectively.
Figure 2:
Schematic diagram of the amino acid
sequence of -tubulin and relevant cleavage sites. FA,
formic acid; CB, cyanogen bromide; GC, endoproteinase
Glu-C; LC, endoproteinase Lys-C.
Figure 3:
HPLC purification by C18 reverse phase
chromatography of -tubulin alkylated with
[
C]3CTC following cyanogen bromide digestion.
Following decylagarose chromatography, 0.25 mg of cyanogen
bromide-digested alkylated
-tubulin was injected onto the HPLC
column, as described in the text. The solid line represents
absorbance and dashed line radiolabel (the maximum reading for
the major radiolabeled peak corresponded to 16,600
cpm).
The C8-repurified peptides underwent sequential Edman degradation.
Material derived from the larger radiolabeled peak of Fig. 3was
subjected to 25 successive cycles, and the results are summarized in Table 1. The analysis indicated that two peptides were present,
and the amino acid recoveries suggest a ratio of about 2 to 1. The
sequence of the predominant peptide, through 17 cycles, was that
expected from cyanogen bromide digestion for the peptide spanning
residues 331-363; and the sequence of the minor peptide, through
10 cycles, for the peptide spanning residues 234-257. Aliquots
from each cycle were analyzed for radiolabel, but recovery of
radioactivity was very low. Increase over background occurred only at
the 6th and 24th cycles (about 11 and 1 pmol, respectively, of
[C] derived from 3CTC), which could correspond
to cysteine residues in the minor and major peptides, respectively (i.e. Cys-239 and Cys-354; see Fig. 2). Material
corresponding to the smaller radiolabeled peak of Fig. 3also
yielded sequence data for peptides 234-257 and 331-363, but
in approximately equal amounts. Radiolabel over background,
representing about 3 pmol of [
C] derived from
3CTC, was recovered in the 6th cycle only. Several attempts to obtain
pure peptides and resolve the apparently paradoxical co-migration of
both peptides in two radiolabeled peaks were unsuccessful.
Figure 4:
Formic
acid digestion of -tubulin alkylated with
[
C]3CTC. Following the 96-h digestion, as
detailed in the text, 20 µg was applied to a 16% polyacrylamide
gel. Electrotransfer to a PVDF membrane was performed, and the membrane
was stained with Coomassie Blue (A), and an autoradiogram of
the membrane was prepared (B). C presents a
densitometry tracing of the autoradiogram. A1 (residues 1-31), A2
(32-304), and A3 (305-445) (nomenclature as in (2) ) are the peptides formed, as shown in Fig. 2, by
cleavage between Asp-31 and Pro-32 and between Asp-304 and
Pro-305.
Following transfer and elution, the radiolabeled A3 band was sequenced, and through 22 cycles the expected amino acids were found (Table 1). In addition, the four most prominent smaller radiolabeled peptides were all found to contain the amino-terminal sequence of the A3 peptide (five cycles of degradation), indicating the secondary hydrolytic sites for formic acid occur primarily in the acidic carboxyl-terminal portion of the A3 peptide.
Insufficient radiolabel was obtained from peptide purification by the gel electrophoresis, membrane transfer, and elution methodology to identify the target amino acid residue in A3 (note, too, that the suspected Cys-354 is 50 residues in from the amino terminus). We therefore decided to explore in situ digestion of A3 (pooled with the A3 fragments) by various agents to further localize the reactive amino acid residue(s).
We began with cyanogen bromide
digestion, to determine whether the results would agree with those
obtained from the partially purified peptide obtained by HPLC.
Digestion of A3 yielded a single radiolabeled peptide (Fig. 5A), and its partial amino acid sequence (Table 1) identified it as the peptide containing residues
331-363. ()
Figure 5:
Autoradiograms of peptides sequenced
following digestion with cyanogen bromide (A), endoproteinase
Glu-C (B), and endoproteinase Lys-C (C). A,
for the cyanogen bromide peptide,
[C]3CTC-peptide A3 was isolated following
polyacrylamide gel electrophoresis and electrotransfer to a
nitrocellulose membrane. The cyanogen bromide digestion was performed in situ on the membrane, and the recovered peptides were
electrophoresed again on a polyacrylamide gel. An electrotransfer to a
PVDF membrane was performed, followed by autoradiography. The
radiolabeled peptide was extracted from the membrane, and its amino
acid sequence (Table 1) was obtained. B and C,
with the endoproteinases, digestion was of
[
C]3CTC-
-tubulin obtained by decylagarose
chromatography. The digests were electrophoresed on polyacrylamide gels
and electrotransferred to PVDF membranes, which were used to prepare
the autoradiograms shown in the figure. The radiolabeled peptides were
extracted from the membranes, and their amino acid sequences (Table 1) were obtained.
In situ digestion of
radiolabeled peptide A3 with endoproteinase Glu-C, which cleaves at the
carboxyl side of glutamate residues, yielded a single radiolabeled band
(data not presented), but we were not successful in isolating enough
material to obtain a definitive amino acid sequence of the peptide. We
therefore treated the [C]3CTC-
-tubulin
complex with the enzyme, but digestion did not appear to be complete,
in that radiolabel was rather diffuse (Fig. 5B).
Nevertheless, there was only one distinct radiolabeled peptide band.
Extraction and sequence analysis (Table 1) demonstrated that it
was the peptide containing residues 344-376, narrowing potential
reactive residue(s) to a somewhat shorter sequence (344-363).
Experiments with endoproteinase Lys-C, which cleaves at the carboxyl
side of lysine residues, were much more successful, yielding, as had
cyanogen bromide digestion of peptide A3, a single prominent
radiolabeled peptide band. As with endoproteinase Glu-C, we were unable
to obtain sufficient peptide for sequencing with in situ digestion of radiolabeled peptide A3, but the -tubulin digest (Fig. 5C) yielded material with the sequence (through
seven cycles) of the peptide containing residues 351-362 (Table 1), further narrowing the possibilities for the reactive
amino acid(s).
As an alternate approach, the endoproteinase Lys-C digest
of the [C]3CTC-
-tubulin was subjected to
automated sequential Edman degradation for twelve cycles (the size of
peptide 351-362), but the entire product stream was collected for
scintillation counting rather than for amino acid analysis (Fig. 6). Some radiolabel was recovered with each cycle, but
only with the fourth cycle was there a dramatic increase in the amount
of radiolabel obtained. Combining this result with the strongly
preferential labeling of peptide 351-362 (Fig. 5C; Table 1), we conclude that the fourth
amino acid of this peptide, Cys-354, is the major target for the
specific covalent interaction of 3CTC with
-tubulin. (There are 15
lysine residues in
-tubulin, and Cys-354 is the only cysteine four
residues carboxyl to a lysine. The other peptides resulting from Lys-C
digestion would have the following amino acids as their fourth residue:
Val-23, Arg-62, Thr-106, Ser-126, Glu-156, Thr-178, Pro-220, Asn-256,
Ala-301, Glu-328, Tyr-340, Thr-366, Glu-383, and His-396.)
Figure 6:
Determination of the radiolabeled amino
acid residue following covalent interaction of
[C]3CTC with
-tubulin. Endoproteinase Lys-C
digestion was performed on the
[
C]3CTC-
-tubulin obtained by decylagarose
chromatography. Material containing approximately 10,000 cpm was
directly subjected to sequential Edman degradation, but the output
stream from each cycle was counted in a liquid scintillation counter
rather than analyzed for its derivatized amino acid content. The
radiolabel recovery from 12 cycles of digestion is
shown.
Attempts to localize the colchicine site of tubulin have
utilized photoaffinity analogs(4, 5) , direct
photoactivation of colchicine(7, 11) , chemically
reactive analogs(17, 18) , and modified tubulin
molecules derived from mutant cells resistant to colchicine site drugs (12, 13, 14) . In the direct approaches, when
the reactive group was in the B ring side chain, the -subunit was
always labeled(5, 7, 17) , although in one
study (5) there was also labeling of
-tubulin. In
contrast, when the tubulin-[
H]colchicine complex
was directly photoactivated at 350 nm, the absorbance maximum
attributable to the tropolone C ring, there was strongly preferential
labeling of
-tubulin(7) . In our own work(18) , we
showed that the chemically reactive thiocolchicine analogs 2CTC and
3CTC, derivatized in the A ring, reacted preferentially with
-tubulin in a reaction that was temperature-dependent and
inhibited by colchicine site drugs.
Uppuluri et al.(11) found that photoactivated colchicine reacted with
amino acid(s) in peptide sequence 1-36 or in peptide sequence
214-241, but not with both peptides. As shown here, 3CTC reacts
strongly with Cys-354 of -tubulin. Since the chloroacetyl group is
relatively small, this indicates that the C-3 oxygen atom of colchicine
is about 3 Å from the sulfur atom of Cys-354. We also found minor
reactivity at a second site. This was most clearly demonstrated in the
large formic acid-derived peptide A2 (residues 32-304). Cyanogen
bromide digestion indicated the second site was in peptide
234-257, and tenuous evidence indicates the second, weakly
reactive amino acid is Cys-239 (within the sequence found by Uppuluri et al.(11) ). (
)
The work of
Ludueña and Roach (27) implicates Cys-354
and Cys-239 in the binding of colchicine to tubulin. The sulfhydryl
cross-linking agent EBI efficiently promoted formation of a cross-link
between these two cysteine residues. EBI, with an ethylene bridge
between two reactive iodoacetamide groups, was the most efficient in a
series of analogs that differed in the size of the spacer, implying the
two sulfur atoms were about 9 Å apart. Formation of the
Cys-239/Cys-354 cross-link is inhibited by every colchicine site agent
examined, except tropolone and tropolone methyl ether, which
may bind weakly to the portion of the tubulin molecule occupied by the
C ring. Following reaction of EBI with tubulin, the protein will no
longer polymerize. ()
Studies with DCBT (28) are also relevant to the present work. This antimitotic compound alkylates multiple cysteine residues of tubulin (29) , but the most reactive, closely associated with DCBT's inhibition of assembly, was Cys-239(20) . Alkylation of Cys-239 by DCBT did not inhibit colchicine binding to tubulin, but colchicine site drugs inhibited alkylation of tubulin by DCBT(20) , analogous to their inhibitory effect on Cys-239/Cys-354 cross-link formation by EBI.
We
also examined -tubulin that had reacted with
2-(chloromethyl-[
C]carbonyl)-2CTC. Following
formic acid digestion radiolabel was distributed in approximately equal
amounts between the A2 and A3 peptides. Further localization of the
radiolabel has been hampered by the 2-4-fold lower covalent
reactivity of 2CTC with
-tubulin. Assuming preferential reactivity
of the chloroacetyl group with cysteine residues (cf. (30, 31, 32) ), this suggests that the C-2
oxygen atom of colchicine is closer to Cys-239 than is the C-3 oxygen,
and that the C-2 oxygen may be equidistant from the Cys-239 and Cys-354
sulfur atoms. The lower reactivity of 2CTC with these cysteines than of
3CTC with Cys-354 indicates that the colchicine C-3 oxygen atom is
closer to Cys-354 than the C-2 oxygen atom is to either cysteine
residue.
Our findings with 3CTC, and to a lesser extent with 2CTC,
suggest that the colchicine A ring lies in the 9 Å pocket between
Cys-354 and Cys-239. Such a model (Fig. 7) is consistent with
the molecular distances obtained from the crystal structure of
colchicine (33) and with the 3 Å length of the
chloroacetyl group. Correlating our findings with those of Uppuluri et al.(11) suggests that, following photoactivation
of colchicine, the reactivity observed with the -subunit sequence
214-241 results from either a direct reaction with the C ring or
energy transfer from the C ring to the A ring, while the reactivity
with the amino-terminal peptide results only from a direct reaction
with the C ring. This would agree with the failure of tropolonic
compounds to inhibit formation of the Cys-239/Cys-354
cross-link(27) .
Figure 7:
A
diagrammatic representation of the proposed interaction of colchicine
with tubulin. The A ring is shown between Cys-354 and Cys-239, while it
is proposed that the tropolone ring interacts with the amino terminus
of -tubulin, to account for the covalent interaction reported by
Uppuluri et al.(11) . The 9-Å distance shown
between the two cysteine sulfur atoms is that postulated to account for
cross-link formation by EBI(27) , and the 3 Å between the
Cys-354 sulfur atom and the C-3 oxygen atom of colchicine is the
calculated length of the chloroacetyl moiety. The intracolchicine
distances were obtained by manipulation of the crystal coordinates in
the Cambridge Structural Data Base (reference code, COLCDH, data from (33) ) with the molecular modeling program Quanta Version 4.0.
The unit cells of the crystal have two molecules of colchicine, and the
two distances shown were nearly identical in both
molecules.
Since the maximum separation of hydrogens
of the C-3 and C-10 methoxy groups is 13.5 Å, this requires the
amino-terminal peptide of tubulin to be relatively close to Cys-239 or
Cys-354. This may well be the case, because one photoaffinity analog of
paclitaxel, modified in its C-13 side chain, reacts covalently with
peptide A1 (residues 1-31) (2) , while another, modified
in the C-2 benzoate moiety of paclitaxel, reacts with the -tubulin
peptide containing residues 217-231(3) . The C-13 side
chain and the C-2 benzoate moiety of taxoids are believed to be closer
to each other in hydrophilic environments (34, 35, 36) than in their crystal
structure(37) . Interhydrogen distances in aqueous solution
below 5 Å between these moieties have been proposed(36) ,
supporting the idea that the amino-terminal peptide must be close to
the peptide spanning amino acids 214-241. These findings also
imply that the binding sites for the C ring of colchicine and for the
interacting C-13/C-2 substituents of paclitaxel may be in close
proximity if not overlapping (cf. (3) ).