©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification of Cysteine 354 of -Tubulin as Part of the Binding Site for the A Ring of Colchicine (*)

(Received for publication, November 16, 1995; and in revised form, February 21, 1996)

Ruoli Bai (1) Xue-Feng Pei (3) Olivier Boyé (2) Zelleka Getahun (1) Surinder Grover (1) Joseph Bekisz (4) Nga Y. Nguyen (4) Arnold Brossi (3) (2) Ernest Hamel (1)(§)

From the  (1)Laboratory of Molecular Pharmacology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, the (2)Laboratory of Structural Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, the (3)Department of Chemistry, Georgetown University, Washington, D. C. 20057, and the (4)Facility for Biotechnology Resources, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-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 beta-tubulin that had reacted with 3-(chloromethyl-[^14C]carbonyl)-3-demethylthiocolchicine ([^14C]3CTC). This beta-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 [^14C]3CTC and beta-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 beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.


INTRODUCTION

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 beta-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 alpha- and beta-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 (^1)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 [^3H]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 beta-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 beta-tubulin occurs primarily at Cys-354.


Figure 1: Separation of the alpha- and beta-subunits of tubulin on decylagarose following interaction of the protein with [^14C]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.




EXPERIMENTAL PROCEDURES

Materials

Preparation of bovine brain tubulin (19) and of 3CTC and [^14C]3CTC (16) were described previously. Specific activity of the [^14C]3CTC was 31 cpm/pmol, and background in the liquid scintillation system used corresponded to about 2 pmol. Decylagarose was from ICN Immunobiologicals; colchicine, cyanogen bromide, PVP40, and NEM from Sigma; ``sequencing grade'' endoproteinase Lys-C (Pseudomonas aeruginosa) from Promega; ``sequencing grade'' endoproteinase Glu-C (Staphylococcus aureus V8) from Boehringer Mannheim; and precast polyacrylamide gels (16% acrylamide), nitrocellulose membranes, and PVDF membranes from Novex.

Preparation of Tubulin Derivatized with 3CTC and Separation of alpha- and beta-Subunits by Decylagarose Chromatography

Reaction mixtures contained 20 µM (2.0 mg/ml) tubulin, 20 µM [^14C]3CTC, 1.0 M monosodium glutamate (2.0 M stock solution adjusted to pH 6.6 with HCl), 0.1 M sodium phosphate (pH 7.0), 0.1 mM GDP, and 0.5 mM MgCl(2). Incubation was for 30 min at 37 °C, and the reaction was stopped by addition of NEM (2.5 mM). The mixture was left overnight at 4 °C to precipitate the tubulin, which was harvested by centrifugation (4000 rpm for 10 min). The pellet was dissolved in Solution A (4 M guanidine HCl and 2 M NaCl at pH 5.0) and applied to a 1.5 times 30-cm column of decylagarose equilibrated with Solution A(20) . The column was washed with Solution A until protein elution ceased, then with 4 M guanidine HCl (pH 5.0). Fractions containing the unbound radiolabeled beta-tubulin (20) were pooled and dialyzed against water, as were the fractions containing the bound alpha-tubulin. Only beta-tubulin of at least 90% purity was used in further studies.

Chemical and Enzymatic Digestions of beta-Tubulin Cross-linked to 3CTC

The [^14C]3CTC-beta-tubulin at 2.0 mg/ml was digested at 37 °C in the dark with either 75% formic acid for 96 h (21) or 20 mg/ml cyanogen bromide in 70% formic acid for 24 h(22) . The samples were frozen, and the formic acid and cyanogen bromide removed under vacuum.

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.

Peptide Purification

HPLC

Preparative HPLC, using an LKB system and Brownlee Laboratories columns, was performed on cyanogen bromide digests of the [^14C]3CTC-beta-tubulin complex. The peptide mixture was dissolved in 4 M guanidine HCl (pH 5.0) and injected onto a C18 Aquapore RP-300 reverse phase column (4.6 times 100 mm). Column output was monitored for absorbance (206 nm) and for radioactivity (Ramona 5-LS flow detector). The column was developed with a 0-60% acetonitrile gradient in 0.1% trifluoroacetic acid. Radioactive peaks were collected and lyophilized. Further HPLC purification was with C8 Aquapore RP-300 CO3-GU and C4 Aquapore BU-300 BO3-GU columns, using, respectively, gradients of 0-30% 2-propanol and 0-60% acetonitrile in 1% trifluoroacetic acid.

Electrophoresis, Electroblotting, and Autoradiography

Peptides were separated on Novex precast Tricine-16% acrylamide polyacrylamide mini-gels (thickness, 1 mm). Peptides were transferred to a PVDF membrane (pore size, 0.2 µm) with an Enprotech transblot system (1.5-2 h, 120 volts). The membrane was stained with Coomassie Blue R-250 and autoradiographed (Kodak BIOMAX MR film; 24-48-h exposure). Radiolabeled peptides were cut from the membrane and eluted with 4 M guanidine HCl (pH 5.0).

In Situ Enzymatic and Chemical Cleavage of Electroblotted Peptides

Following electrophoresis on the Tricine-16% acrylamide gel, peptides were electroblotted to a nitrocellulose membrane. This was stained with Ponceau S and autoradiographed. Equivalent radiolabeled peptide bands were cut from several membranes, destained, pooled in a single vessel, and incubated for 30 min at 37 °C in 3 ml of 0.5% PVP40 in 0.1 M acetic acid, which prevents adsorption of cyanogen bromide or protease to the membrane. Excess PVP40 was removed with water. The radioactive strips were cut into 1-mm squares and digested with cyanogen bromide, endoproteinase Lys-C, or endoproteinase Glu-C as described above. Peptides released from the membrane squares were again electrophoresed on Tricine-16% acrylamide gels, electroblotted to PVDF membranes, autoradiographed, and eluted with 4 M guanidine HCl.

Sequence Analysis

Automated Edman degradation for determination of amino acid sequence was performed with an Applied Biosystems model 470A gas-phase sequencer(24) . Identification of phenylthiohydantoin amino acid derivatives was carried out with an Applied Biosystems model 120 PTH Analyzer(25) .


RESULTS

Isolation of Covalently Modified beta-Tubulin

We initially prepared a reaction mixture containing equimolar concentrations of tubulin and [^14C]3CTC. NEM was used to irreversibly alkylate cysteine residues and precipitate the tubulin, permitting separation of unreacted drug and drug-protein complex by centrifugation.

Denatured tubulin subunits can be resolved by hydrophobic chromatography on decylagarose(20) . Fig. 1summarizes a separation following reaction with [^14C]3CTC. In the presence of 2 M NaCl, 4 M guanidine HCl the column only binds alpha-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 beta-tubulin. In the preparation shown in Fig. 1, there was over five times as much radiolabel associated with beta-tubulin as with alpha-tubulin, with drug/monomer stoichiometries of 0.32 and 0.06, respectively.

Overview of Pertinent Cleavage Sites in the 3CTC-beta-Tubulin Complex

Fig. 2presents a diagram of the chemical and protease cleavage sites used to define the covalent interaction of 3CTC with beta-tubulin (complete amino acid sequence in (26) ). With formic acid, cleavage occurs preferentially between aspartate and proline residues(21) , and two such sites are present in beta-tubulin. We found minor incorporation of radiolabel into the peptide spanning residues 32-304 and major incorporation into the carboxyl-terminal peptide (residues 305-445). Cyanogen bromide cleaves polypeptides on the carboxyl-terminal side of methionine residues(22) . The cyanogen bromide studies were consistent with incorporation of radiolabel both at Cys-239 and in a peptide spanning residues 331-363. Radiolabel was found predominantly in a peptide whose sequence was consistent with residues 344-376 following digestion with endoproteinase Glu-C and with residues 351-362 following digestion with endoproteinase Lys-C. These proteases cleave at the carboxyl end of the indicated amino acids (23) . Insufficient quantities of these peptides were available for identification of the radiolabeled amino acid. Sequential Edman degradation was performed on the entire beta-tubulin digest following digestion with endoproteinase Lys-C, and evaluation of radiolabel as opposed to sequence analysis was performed following each cycle. Maximum recovery of radiolabel occurred after the fourth cycle, confirming the conclusion that Cys-354 was the amino acid residue that preferentially reacted with [^14C]3CTC.


Figure 2: Schematic diagram of the amino acid sequence of beta-tubulin and relevant cleavage sites. FA, formic acid; CB, cyanogen bromide; GC, endoproteinase Glu-C; LC, endoproteinase Lys-C.



Sequence Analysis of HPLC-purified Cyanogen Bromide Digests of the 3CTC-beta-Tubulin Complex

Our initial approach was to digest the purified [^14C]3CTC-beta-tubulin complex with cyanogen bromide and isolate radiolabeled peptide(s) by preparative HPLC on a reverse phase C18 column (Fig. 3). With digestion times of 24-72 h we obtained two radiolabeled peaks, with the first peak two to three times larger than the second. With shorter digestion times the second peak was significantly larger, suggesting it was a precursor of the first. Repurification of the first peak on a reverse phase C8 column produced a sharp radiolabeled peak, but upon repurification the second peak was broad (data not presented). Further purification on a reverse phase C4 column was not successful.


Figure 3: HPLC purification by C18 reverse phase chromatography of beta-tubulin alkylated with [^14C]3CTC following cyanogen bromide digestion. Following decylagarose chromatography, 0.25 mg of cyanogen bromide-digested alkylated beta-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 [^14C] 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 [^14C] 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.



Microsequence Analysis of Peptides Purified by SDS-Polyacrylamide Gel Electrophoresis

We next turned to formic acid digestion(21) . In this and subsequent studies radiolabeled peptides were resolved by polyacrylamide gel electrophoresis, with electrophoretic transfer of the separated peptides to PVDF or nitrocellulose membranes, and the peptides were located by autoradiography. As noted by Rao et al.(2) , beta-tubulin has only two major formic acid cleavage sites (between aspartate and proline residues), at positions 31/32 and 304/305. This results in three peptides of highly divergent molecular mass: the amino-terminal A1, spanning residues 1-31, and the much larger A2(32-304) and A3(305-445). In our hands formic acid digests of the [^14C]3CTC-beta-tubulin complex yielded major peptide bands with molecular weights consistent with the expected products (Fig. 4A) and multiple minor bands. With shorter incubations these minor bands were less prominent, but residual beta-tubulin persisted, and there were other higher molecular weight bands. Autoradiography of the digested beta-tubulin (Fig. 4B) showed significant radiolabel in six peptides, A2, A3, and four smaller species. Densitometry of the autoradiogram (Fig. 4C) demonstrated that less than 10% of the radiolabel was in the A2 band, consistent with a weak covalent interaction of [^14C]3CTC with Cys-239, but this peptide was not evaluated further.


Figure 4: Formic acid digestion of beta-tubulin alkylated with [^14C]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. (^2)


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, [^14C]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 [^14C]3CTC-beta-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 [^14C]3CTC-beta-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 beta-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).

Identification of Cys-354 as the Amino Acid Residue of beta-Tubulin That Reacts with 3CTC

Although sufficient amounts of radiolabeled peptide could be extracted from the gels for sequencing, the amount of radiolabel was insufficient for defining the reactive amino acid(s). We had also obtained only suggestive information from the partially purified HPLC cyanogen bromide peptides when sequencing and radiolabel data were obtained from the same Edman digestion.

As an alternate approach, the endoproteinase Lys-C digest of the [^14C]3CTC-beta-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 beta-tubulin. (There are 15 lysine residues in beta-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 [^14C]3CTC with beta-tubulin. Endoproteinase Lys-C digestion was performed on the [^14C]3CTC-beta-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.




DISCUSSION

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 alpha-subunit was always labeled(5, 7, 17) , although in one study (5) there was also labeling of beta-tubulin. In contrast, when the tubulin-[^3H]colchicine complex was directly photoactivated at 350 nm, the absorbance maximum attributable to the tropolone C ring, there was strongly preferential labeling of beta-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 beta-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 beta-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) ). (^3)

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. (^4)

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 beta-tubulin that had reacted with 2-(chloromethyl-[^14C]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 beta-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 beta-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 beta-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 beta-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) ).


FOOTNOTES

*
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: Bldg. 37, Rm. 5C25, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-4855; Fax: 301-496-5839.

(^1)
The abbreviations used are: 2CTC, 2-chloroacetyl-2-demethylthiocolchicine; 3CTC, 3-chloroacetyl-3-demethylthiocolchicine; [^14C]3CTC, 3-(chloromethyl-[^14C]carbonyl)-3-demethylthiocolchicine; PVP40, polyvinylpyrrolidone (average molecular mass, 40 kDa); NEM, N-ethylmaleimide; HPLC, high-performance liquid chromatography; PVDF, polyvinylidene difluoride; DCBT, 2,4-dichlorobenzyl thiocyanate; EBI, N,N`-ethylenebis(iodoacetamide); Tricine, N-[2-hydroxy-1,1bis(hydroxymethyl)ethyl]glycine.

(^2)
The radiolabeled peptides generated by cyanogen bromide and endoproteinase digestion of either [^14C]3CTC-A3 or [^14C]3CTC-beta-tubulin did not correspond to well defined Coomassie Blue-stained bands, suggesting that the modified and unmodified peptides have significantly different electrophoretic mobilities (maximum stoichiometry of the total covalent reaction, about 0.3).

(^3)
Cys-354 is present in all beta-tubulin isotypes found in brain, while position 239 is 75% cysteine (three isotypes) and 25% serine (one isotype)(27) .

(^4)
Ludueña and Roach (27) have also shown that EBI will cross-link Cys-12 with either Cys-201 or Cys-211 of beta-tubulin, indicating a 9-Å distance between the two sulfur atoms involved.


ACKNOWLEDGEMENTS

We thank J. Tropea, University of Michigan, for performing most of the peptide sequence determinations and Dr. Marc Nicklaus, National Cancer Institute, for assistance with determining the intramolecular distances in colchicine from the crystal data.


REFERENCES

  1. Nogales, E., Wolf, S. G., Khan, I. A., Ludueña, R. F., and Downing, K. H. (1995) Nature 375, 424-427 [CrossRef][Medline] [Order article via Infotrieve]
  2. 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 [Abstract/Free Full Text]
  3. Rao, S., Orr, G. A., Chaudhary, A. G., Kingston, D. G. I., and Horwitz, S. B. (1995) J. Biol. Chem. 270, 20235-20238 [Abstract/Free Full Text]
  4. Williams, R. F., Mumford, C. L., Williams, G. A., Floyd, L. J., Aivaliotis, M. J., Martinez, R. A., Robinson, A. K., and Barnes, L. D. (1985) J. Biol. Chem. 260, 13794-13802 [Abstract/Free Full Text]
  5. Floyd, L. J., Barnes, L. D., and Williams, R. F. (1989) Biochemistry 28, 8515-8525 [Medline] [Order article via Infotrieve]
  6. Shivanna, B. D., Mejillano, M. R., Williams, T. D., and Himes, R. H. (1993) J. Biol. Chem. 268, 127-132 [Abstract/Free Full Text]
  7. Wolff, J., Knipling, L., Cahnmann, H. J., and Palumbo, G. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2820-2824 [Abstract]
  8. Rao, S., Horwitz, S. B., and Ringel, I. (1992) J. Natl. Cancer Inst. 84, 785-788 [Abstract]
  9. Hastie, S. B. (1991) Pharmacol. & Ther. 51, 377-401
  10. Weisenberg, R. C., Borisy, G. G., and Taylor, E. W. (1968) Biochemistry 7, 4466-4477 [Medline] [Order article via Infotrieve]
  11. Uppuluri, S., Knipling, L., Sackett, D. L., and Wolff, J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11598-11602 [Abstract]
  12. Keates, R. A. B., Sarangi, F., and Ling, V. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5638-5642 [Abstract]
  13. Cabral, F., Sobel, M. E., and Gottesman, M. M. (1980) Cell 20, 29-36 [CrossRef][Medline] [Order article via Infotrieve]
  14. Sheir-Neiss, G., Lai, M. H., and Morris, N. R. (1978) Cell 15, 639-647 [Medline] [Order article via Infotrieve]
  15. Boyé, O., Hamel, E., and Brossi, A. (1991) Med. Chem. Res. 1, 142-150
  16. Boyé, O., Getahun, Z., Grover, S., Hamel, E., and Brossi, A. (1992) J. Labelled Compounds Radiopharm. 33, 293-299
  17. Schmitt, H., and Atlas, D. (1976) J. Mol. Biol. 102, 743-758 [Medline] [Order article via Infotrieve]
  18. Grover, S., Boyé, O., Getahun, Z., Brossi, A., and Hamel, E. (1992) Biochem. Biophys. Res. Commun. 187, 1350-1358 [Medline] [Order article via Infotrieve]
  19. Hamel, E., and Lin, C. M. (1984) Biochemistry 23, 4173-4184 [Medline] [Order article via Infotrieve]
  20. Bai, R., Lin, C. M., Nguyen, N. Y., Liu, T.-Y., and Hamel, E. (1989) Biochemistry 28, 5606-5612 [Medline] [Order article via Infotrieve]
  21. Landon, M. (1977) Methods Enzymol. 47, 145-149 [Medline] [Order article via Infotrieve]
  22. Gross, E., and Witkop, B. (1961) J. Am. Chem. Soc. 83, 1510-1511
  23. Wilkinson, J. M. (1986) in Practical Protein Chemistry: A Handbook (Darbre, A., ed) pp. 122-148, John Wiley and Sons, New York
  24. Hewick, R. M., Hunkapiller, M. W., Hood, L. E., and Dryck, W. J. (1981) J. Biol. Chem. 256, 7990-7997 [Abstract/Free Full Text]
  25. Zimmerman, C. L., Appella, E., and Pisano, J. J. (1977) Anal. Biochem. 77, 569-573 [Medline] [Order article via Infotrieve]
  26. Krauhs, E., Little, M., Kempf, T., Hofer-Warbinek, R., Ade, W., and Ponstingl, H. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 4156-4160 [Abstract]
  27. Ludueña, R. F., and Roach, M. C. (1991) Pharmacol. & Ther. 49, 133-152
  28. Abraham, I., Dion, R. L., Duanmu, C., Gottesman, M. M., and Hamel, E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6839-6843 [Abstract]
  29. Bai, R., Duanmu, C., and Hamel, E. (1989) Biochim. Biophys. Acta 994, 12-20 [Medline] [Order article via Infotrieve]
  30. Schmitt, H., and Kram, R. (1978) Exp. Cell Res. 115, 408-411 [Medline] [Order article via Infotrieve]
  31. Biellmann, J. F., Branlant, G., Nicolas, J.-C., Pons, M., Descomps, B., and Crastes de Paulet, A. (1976) Eur. J. Biochem. 63, 477-481 [Abstract]
  32. McCarthy, A. D., and Hardie, D. G. (1982) FEBS Lett. 147, 256-260 [CrossRef][Medline] [Order article via Infotrieve]
  33. Lessinger, L., and Margulis, T. N. (1978) Acta Crystallogr. B34, 578-584
  34. Williams, H. J., Scott, I., Dieden, R. A., Swindell, C. S., Chirlian, L. E., Francl, M. M., Heerding, J. M., and Krauss, N. E. (1993) Tetrahedron 49, 6545-6560 [CrossRef]
  35. Dubois, J., Guénard, D., Guéritte-Voegelein, F., Guedira, N., Potier, P., Gillet, B., and Beloeil, J.-C. (1993) Tetrahedron 49, 6533-6544 [CrossRef]
  36. Vander Velde, D. G., Georg, G. I., Grunewald, G. L., Gunn, C. W., and Mitscher, L. A. (1993) J. Am. Chem. Soc. 115, 1650-1651
  37. Guéritte-Voegelein, F., Guénard, D., Mangatal, L., Potier, P., Guilhem, J., Cesario, M., and Pascard, C. (1990) Acta Crystallogr. C46, 781-784

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