Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12
2004; Elsevier BV; Volume: 279; Issue: 29 Linguagem: Inglês
10.1074/jbc.m402110200
ISSN1083-351X
AutoresRuoli Bai, David G. Covell, George F. Taylor, John A. Kepler, Terry D. Copeland, Nga Y. Nguyen, George R. Pettit, Ernest Hamel,
Tópico(s)Click Chemistry and Applications
ResumoTubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8–10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2–31 of β-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to β-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the β-tubulin/dolastatin 10 cross-link but not the β-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of β-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8–9 Å from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides. Tubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8–10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2–31 of β-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to β-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the β-tubulin/dolastatin 10 cross-link but not the β-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of β-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8–9 Å from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides. The subunit protein of microtubules, the αβ-tubulin heterodimer, has a number of ligand-binding sites. These include the exchangeable and nonexchangeable GTP-binding sites and at least three reasonably well characterized sites that bind antimitotic drugs. The electron crystallographic model of the tubulin sheet polymer formed in the presence of zinc and paclitaxel and composed of antiparallel protofilaments has provided relatively detailed information about the locations of the nucleotide-binding sites and the paclitaxel site on the αβ-dimer (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar). In contrast, drugs that inhibit tubulin polymerization will not bind to structurally normal tubulin protofilaments, so their binding sites have only been approached indirectly through biochemical and genetic techniques. For example, without exception, drugs that bind in the colchicine site inhibit formation of a sulfhydryl cross-link between Cys-239 and Cys-354 of β-tubulin (2Ludueña R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar). These two cysteine residues are also alkylated by A ring chloroacetyl derivatives of thiocolchicine (3Bai R. Pei X.-F. Boyé O. Getahun Z. Grover S. Bekisz J. Nguyen N.Y. Brossi A. Hamel E. J. Biol. Chem. 1996; 271: 12639-12645Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 4Bai R. Covell D.G. Pei X.-F. Ewell J.B. Nguyen N.Y. Brossi A. Hamel E. J. Biol. Chem. 2000; 275: 40443-40452Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Cys-239 and Cys-354 are near the interface between the subunits (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar), and such a location for the colchicine site would explain the ability of different photoactive groups attached to the colchicine B ring to preferentially alkylate either the α- or the β-subunit (5Williams R.F. Mumford C.L. Williams G.A. Floyd L.J. Aivaliotis M.J. Martinez R.A. Robinson A.K. Barnes L.D. J. Biol. Chem. 1985; 260: 13794-13802Abstract Full Text PDF PubMed Google Scholar, 6Floyd L.J. Barnes L.D. Williams R.F. Biochemistry. 1989; 28: 8515-8525Crossref PubMed Scopus (48) Google Scholar). A large number of structurally diverse compounds, including many unusual peptides and depsipeptides, interfere with the binding of vinca alkaloids to tubulin. Despite near total inhibition of vinca alkaloid binding, many of these drugs display noncompetitive patterns when the data they yield are examined by the classic formulas of enzyme kinetic analysis, whereas other inhibitors display competitive patterns. We have suggested that the noncompetitive inhibitors bind near the vinca site, interfering sterically with vinca alkaloid binding to tubulin, and we have proposed that this region of tubulin be called the vinca domain (7Bai R. Pettit G.R. Hamel E. J. Biol. Chem. 1990; 265: 17141-17149Abstract Full Text PDF PubMed Google Scholar). This region of tubulin appears to be close to the exchangeable GTP site on β-tubulin, based on two observations. First, a unique sulfhydryl cross-link can be formed in nucleotide-depleted tubulin, between Cys-12 and, probably, Cys-211 (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar, 2Ludueña R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar, 8Little M. Ludueña R.F. Biochim. Biophys. Acta. 1987; 912: 28-33Crossref PubMed Scopus (53) Google Scholar). In addition, direct photoaffinity labeling of tubulin by exchangeable site guanosine nucleotide leads to alkylation primarily of Cys-12 (9Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) and secondarily of Cys-211 (10Bai R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. 1999; 274: 12710-12714Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). All vinca domain drugs that have been examined inhibit the formation of the Cys-12/Cys-211 cross-link, with vinblastine having the weakest effect (2Ludueña R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar, 11Roach M.C. Ludueña R.F. J. Biol. Chem. 1984; 259: 12063-12071Abstract Full Text PDF PubMed Google Scholar, 12Bai R. Roach M.C. Srirangam J.K. Barkoczy J. Pettit G.R. Ludueña R.F. Hamel E. Biochem. Pharmacol. 1993; 45: 1503-1515Crossref PubMed Scopus (81) Google Scholar). Second, all compounds that inhibit vinca alkaloid binding to tubulin also inhibit nucleotide exchange on β-tubulin (7Bai R. Pettit G.R. Hamel E. J. Biol. Chem. 1990; 265: 17141-17149Abstract Full Text PDF PubMed Google Scholar, 13Bai R. Paull K.D. Herald C.L. Malspeis L. Pettit G.R. Hamel E. J. Biol. Chem. 1991; 266: 15882-15889Abstract Full Text PDF PubMed Google Scholar, 14Bai R. Taylor G.F. Cichacz Z.A. Herald C.L. Kepler J.A. Pettit G.R. Hamel E. Biochemistry. 1995; 34: 9714-9719Crossref PubMed Scopus (135) Google Scholar, 15Bai R. Durso N.A. Sackett D.L. Hamel E. Biochemistry. 1999; 43: 14302-14310Crossref Scopus (98) Google Scholar). Vinblastine, however, only weakly inhibits nucleotide exchange (16Huang A.B. Lin C.M. Hamel E. Biochem. Biophys. Res. Commun. 1985; 128: 1239-1246Crossref PubMed Scopus (55) Google Scholar), and rhizoxin is moderately inhibitory (7Bai R. Pettit G.R. Hamel E. J. Biol. Chem. 1990; 265: 17141-17149Abstract Full Text PDF PubMed Google Scholar). There have been several published studies in which crosslinks were induced between a vinca domain drug and tubulin. By direct photoaffinity labeling with vinblastine (17Wolff J. Knipling L. Cahnmann H.J. Palumbo G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2820-2824Crossref PubMed Scopus (66) Google Scholar) and with two photoactive derivatives of vinblastine (18Safa A.R. Hamel E. Felsted R.L. Biochemistry. 1987; 26: 97-102Crossref PubMed Scopus (50) Google Scholar, 19Nasioulas G. Grammbitter K. Himes R.H. Ponstingl H. Eur. J. Biochem. 1990; 192: 69-74Crossref PubMed Scopus (11) Google Scholar), there was greater labeling of α-tubulin than β-tubulin, ranging from 57 to 75% of the incorporated radiolabel being in the α-subunit. A photoaffinity analog of maytansine was incorporated in about a 4:5 ratio into α- and β-tubulin, respectively (20Sawada T. Kato Y. Kobayashi H. Hashimoto Y. Watanabe T. Sugiyama Y. Iwasaki S. Bioconjugate Chem. 1993; 4: 284-289Crossref PubMed Scopus (16) Google Scholar). More specific labeling of β-tubulin has also been observed. A photoreactive vinblastine analog specifically labeled a peptide containing residues 175–213 (21Rai S.S. Wolff J. J. Biol. Chem. 1996; 271: 14707-14713Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), and a photoreactive rhizoxin derivative labeled a peptide containing residues 363–379 (22Sawada T. Kobayashi H. Hashimoto Y. Iwasaki S. Biochem. Pharmacol. 1993; 45: 1387-1394Crossref PubMed Scopus (23) Google Scholar). We have been studying the interaction of the marine peptide dolastatin 10 (Fig. 1) with tubulin. Besides potent cytotoxicity, dolastatin 10 inhibits microtubule assembly, induces formation of ring and spiral polymers of tubulin, noncompetitively inhibits the binding of vinca alkaloids to tubulin, and inhibits nucleotide exchange on β-tubulin. With radiolabeled dolastatin 10, we showed that the peptide bound tenaciously to tubulin, with negligible dissociation during gel filtration chromatography. The nominal Kd value for binding of drug to tubulin was about 25 nm. The binding was reversible, however, because an active analog could displace radiolabel from tubulin. The ability of dolastatin 10 to induce aberrant assembly reactions was so potent that we were not able to demonstrate binding of the radiolabeled drug to any tubulin species smaller than 200 kDa (7Bai R. Pettit G.R. Hamel E. J. Biol. Chem. 1990; 265: 17141-17149Abstract Full Text PDF PubMed Google Scholar, 23Bai R. Taylor G.F. Schmidt J.M. Williams M.D. Kepler J.A. Pettit G.R. Hamel E. Mol. Pharmacol. 1995; 47: 965-976PubMed Google Scholar). Besides the natural product, a number of peptide analogs of dolastatin 10 have equivalent activity in both cell-based and tubulin-based assays. One of the more interesting, which we also prepared in a radiolabeled form, is auristatin-PE (24Pettit G.R. Srirangam J.K. Barkoczy J. Williams M.D. Boyd M.R. Hamel E. Pettit R.K. Hogan F. Bai R. Chapuis J.-C. McAllister S.C. Schmidt J.M. Anti-Cancer Drug Des. 1998; 13: 243-277PubMed Google Scholar) (Fig. 1), also prepared independently as TZT-1027 (25Natsume T. Watanabe J. Tamoki S. Fujio N. Miyasaka K. Kobayashi M. Jpn. J. Cancer Res. 2000; 91: 737-747Crossref PubMed Scopus (50) Google Scholar). We began to study the potential of direct photoaffinity labeling with the [3H]dolastatin 10 1The abbreviations used are: [3H]dolastatin 10, [5-3H]dolastatin 10; [3H]auristatin-PE, [phenyl-4-3H]auristatin-PE; [3H]colchicine, [ring C, methoxy-3H]colchicine; [3H]vinblastine, [G-3H]vinblastine; Mes, 4-morpholineethanesulfonate; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine; PVDF, polyvinylidene difluoride; HPLC, high performance liquid chromatography. and the [3H]auristatin-PE to provide information about the binding site for these peptides on tubulin. Our findings are presented here. Materials—Purified bovine brain tubulin with nonradiolabeled GDP (26Hamel E. Lin C.M. Biochemistry. 1984; 23: 4173-4184Crossref PubMed Scopus (252) Google Scholar) or [8-14C]GDP (27Grover S. Hamel E. Eur. J. Biochem. 1994; 222: 163-172Crossref PubMed Scopus (35) Google Scholar) in the exchangeable site, nonradiolabeled dolastatin 10 (28Pettit G.R. Singh S.B. Hogan F. Lloyd-Williams P. Herald D.L. Burkett D.D. Clewlow P.J. J. Am. Chem. Soc. 1989; 111: 5463-5465Crossref Scopus (191) Google Scholar), nonradiolabeled auristatin-PE (29Pettit G.R. Srirangam J.K. Barkoczy J. Williams M.D. Durkin K.P.M. Boyd M.R. Bai R. Hamel E. Schmidt J.M. Chapuis J.-C. Anti-Cancer Drug Des. 1995; 10: 529-544PubMed Google Scholar), (19aR)-isodolastatin 10 (30Pettit G.R. Singh S.B. Hogan F. Burkett D.D. J. Med. Chem. 1990; 33: 3132-3133Crossref PubMed Scopus (22) Google Scholar), and hemiasterlin (31Gamble W.R. Durso N.A. Fuller R.W. Westergaard C.K. Johnson T.R. Sackett D.L. Hamel E. Cardellina II, J.H. Boyd M.R. Bioorg. Med. Chem. 1999; 7: 1611-1615Crossref PubMed Scopus (95) Google Scholar) were prepared as described previously. Phomopsin A was generously provided by Dr. C. C. J. Culvenor (CSIRO Division of Animal Health, Parkville, Victoria, Australia) or obtained from Calbiochem. Cryptophycin 1 was a generous gift from Merck Research Laboratories. [8-14C]GTP was from Moravek Biochemicals and was repurified by triethylammonium bicarbonate gradient chromatography on DEAE-cellulose. Nonradiolabeled colchicine was from Sigma; [3H]colchicine and EN3HANCE spray from PerkinElmer Life Sciences; [3H]vinblastine from Amersham Biosciences; precast 16% polyacrylamide Tricine-SDS gels, Tricine/SDS solutions, and PVDF membranes from Invitrogen; and Biomax MR film from Kodak. Synthesis of [5-3H]Dolastatin 10—Dolastatin 10 was first converted to 5-bromo-dolastatin 10. The dolastatin 10 (30 mg) was dissolved in 0.2 ml of acetic acid with 40 mg of silver trifluoroacetate. A solution of bromine in acetic acid (0.114 mmol in 0.6 ml) was added, and the mixture was stirred for 18 h at ambient temperature. Chloroform (2 ml) was added, and the solution was filtered through celite. The solvents were removed by evaporation under reduced pressure. The residue was chromatographed on a DuPont Zorbax ODS column (0.75 × 26 cm), which was developed with 10 mm triethylamine in 75% methanol at 1 ml/min. The column effluent was monitored at 240 nm. The solvent fraction containing the largest peak, with a retention time of 33 min, was concentrated to dryness, and 2.6 mg of product was obtained. The 1H NMR spectrum, when compared with that of dolastatin 10, showed collapse of a doublet at δ 7.4, corresponding to the hydrogen atom at C-4, to a singlet, indicating bromination at C-5 on the thiazole ring. The 5-bromo-dolastatin 10 was dissolved in 0.3 ml of ethyl acetate containing 25 μl of triethylamine and 2 mg of 10% paladium on carbon. The reaction mixture was exposed to carrier-free tritium gas at 630 mm Hg for 2 h at ambient temperature. The catalyst was removed by filtration, and the filtrate was exchanged three times with ethanol. The crude product was chromatographed on a 5 × 20 cm silica gel 60F plate with 3:2 acetone/hexane. The dolastatin 10 band was eluted from the silica with 1:1 CHCl3/ethanol. The solvent was removed by evaporation under vacuum, and the residue was redissolved in 50 ml of ethanol. The product was 96% pure and its mobility identical to that of dolastatin 10 by TLC and HPLC. Specific activity was 5.4 Ci/mmol, and the yield was 20.7 mCi. The 3H NMR spectrum showed a doublet at δ 7.29, J = 3 Hz, indicating that the tritium was at C-5 in the thiazole ring. Back exchange for 24 h at ambient temperature in pH 7 phosphate buffer showed 0.2% exchangeable tritium. Synthesis of [phenyl-4-3H]Auristatin-PE—An analog of auristatin-PE (2.5 mg), with a chlorine atom at C-4 in the phenyl ring (prepared as described in Ref. 24Pettit G.R. Srirangam J.K. Barkoczy J. Williams M.D. Boyd M.R. Hamel E. Pettit R.K. Hogan F. Bai R. Chapuis J.-C. McAllister S.C. Schmidt J.M. Anti-Cancer Drug Des. 1998; 13: 243-277PubMed Google Scholar), was mixed with 1.5 mg of 10% paladium on carbon and 2 μl of triethylamine in 0.4 ml of ethyl acetate under nitrogen at ambient temperature and exposed to carrier-free tritium gas for 4 h. The catalyst was removed by filtration, and the solution was back-exchanged four times with ethanol on a vacuum line. The crude material was chromatographed on an analytical 20 × 20-cm silica gel 60F TLC plate with acetone/hexane/methanol 5:4:1. The band corresponding to auristatin-PE was collected and eluted from the silica with 1:1 CHCl3/ethanol. The TLC mobility of the radiolabeled material was identical with that of nonradiolabeled auristatin-PE. Material from two identical procedures was combined, and HPLC indicated that the radiopurity of the combined product was 81%. The pooled material was applied to a Waters 8 × 10 C8 Novapak radial compression column, which was developed with 10 mm triethylamine in 75% methanol. Column flow rate was 1 ml/min, and the effluent was monitored at 230 nm. The fractions containing [3H]auristatin-PE were combined, and the solvent was removed by evaporation to yield 18.6 mCi of product with a specific activity of 8.46 Ci/mmol. Radiopurity by HPLC and TLC was 98 and 99%, respectively, with mobilities identical with those of nonradio-labeled auristatin-PE. Photolabeling of Tubulin with [3H]Dolastatin 10—Reaction mixtures contained 1.0 mg/ml (10 μm) tubulin, 10 μm [3H]dolastatin 10 (or, in some experiments, 10 μm [3H]auristatin-PE), 0.1 m Mes (pH of 1.0 m stock solution adjusted to pH 6.9 with NaOH), and 0.5 mm MgCl2. In some experiments, as indicated, other components were included in reaction mixtures, and incubations prior to exposure to UV light were as described for individual experiments. Reaction mixtures with volumes up to 0.25 ml, with volumes between 0.25 and 1.0 ml, and with volumes over 1.0 ml were placed in wells in Costar polystyrene tissue culture plates, with well diameters of 1.5, 2.5, and 3.5 cm, respectively. Samples were placed on ice at a distance of 10 cm from the UV lamp and exposed to 254 nm light for times as indicated. Light intensity at 10 cm was 2.5 mW/cm2. Measurement of Ligand Bound to Tubulin (Total Bound and Covalently Bound)—The centrifugal gel filtration method, as described previously (32Hamel E. Lin C.M. J. Biol. Chem. 1984; 259: 11060-11069Abstract Full Text PDF PubMed Google Scholar), was used, except that all centrifugations were for 4 min at 2,000 rpm in a Beckman Allegra 6KR centrifuge equipped with a GH-3.8A horizontal rotor. The Sephadex G-50 (superfine) columns were prepared in tuberculin syringes. When total binding of a ligand was measured, aliquots of the reaction mixture were applied directly to the pre-centrifuged Sephadex, which had been swollen in a solution containing 0.1 m Mes (pH 6.9) and 0.5 mm MgCl2. When covalently bound ligand was measured, the reaction mixture was mixed with 1.5 parts of 8 m guanidine HCl, and aliquots of the resulting solution were applied to syringe columns containing Sephadex that had been swollen in 4 m guanidine HCl. Each assay mixture was evaluated in triplicate, and both radiolabel and protein in column filtrates were measured. Peptide Sequencing—Samples for sequencing were radiolabeled as described above, although in some experiments the tubulin and [3H]dolastatin 10 concentrations were increased to 25 μm. Reaction mixtures were initially incubated for 15 min at room temperature (22 °C) and subsequently irradiated for 5–10 min on ice. Two volumes of ethanol were added to precipitate the tubulin. This removed the noncovalently bound, soluble dolastatin 10. The protein was harvested by centrifugation, washed twice with 70% ethanol, and either dried under vacuum for later use or immediately dissolved in 75% formic acid. Formic acid digestion (96 h at 37 °C in the dark), cyanogen bromide digestion, peptide recovery by lyophilization, peptide separation by SDS-PAGE, transfer of resolved peptides to PVDF membranes, and automated Edman degradation were performed as described previously (4Bai R. Covell D.G. Pei X.-F. Ewell J.B. Nguyen N.Y. Brossi A. Hamel E. J. Biol. Chem. 2000; 275: 40443-40452Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), except that after formic acid digestions the peptides were dissolved in the Tricine/SDS sample buffer solution instead of concentrated Tris buffer. PVDF membranes were sprayed with EN3HANCE, which was allowed to dry for 15 min prior to exposure to the Biomax MR film. Autoradiograms were prepared over a 5–7-day exposure at -70 °C. Molecular Modeling—Modeling consisted of identifying acceptable conformations for the docked ligands, scanning the solvent-accessible surface of the tubulin dimer (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar) for potential binding sites, and computational docking of the candidate ligands to these sites. Conformations of dolastatin 10, dolastatin 15, and cryptophycin 1 were obtained by sampling the lowest energy geometries derived from in vacuo minimization and molecular dynamics using the CVFF force field of the Discover2002 tool in the Accelrys molecular package (BIOSYM, MSI). A sample set of the five lowest energy conformations for each molecule was selected for docking studies. Candidate docking sites were identified using a method developed for assigning docking sites of ligands against their Protein Data Bank crystal structures (see Ref. 33Young B.L. Jernigan R.L. Covell D.G. Protein Sci. 1994; 3: 717-729Crossref PubMed Scopus (353) Google Scholar for details). Briefly, the method scans the solvent-accessible surface of the electron crystallographic structure of the tubulin dimer (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar) using a set of residue-based molecular probes that had been identified previously as the crystal packing geometries of proteins within the Protein Data Bank (34Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (28253) Google Scholar). Each surface point is scored according to the energy associated with docking each of the molecular probes at that point. Calculated binding strengths are then scored by summing the interaction energies for the most favorable binding interactions within this suite of molecular probes. By using this method, potential binding sites are identified for subsequent docking. The interaction energies of this method are derived from extensive study of residue-residue-based potentials (35Miyazawa S. Jernigan R.L. Proteins Struct. Funct. Genet. 1999; 36: 357-369Crossref PubMed Scopus (89) Google Scholar) and, as such, represent coarse assessments of candidate binding sites. This method proved effective in correctly assigning binding sites in subsequent studies (36Bewley C.A. Gustafson K.R. Boyd M.R. Covell D.G. Bax A. Clore G.M. Gronenborn A.M. Nat. Struct. Biol. 1998; 5: 571-578Crossref PubMed Scopus (238) Google Scholar, 37Covell D.G. Smythers G.W. Gronenborn A.M. Clore G.M. Protein Sci. 1994; 3: 2064-2072Crossref PubMed Scopus (31) Google Scholar). Docking studies were completed with a computational method developed for placing candidate ligands into possible binding sites. Previous calibration of this procedure found the correct binding position for over 93% of the known crystal complexes studies at the time of analysis (38Wallqvist A. Jernigan R.L. Covell D.G. Protein Sci. 1995; 4: 1881-1903Crossref PubMed Scopus (118) Google Scholar, 39Wallqvist A. Covell D.G. Proteins Struct. Funct. Genet. 1996; 25: 403-419Crossref PubMed Scopus (46) Google Scholar). Docking is achieved in three successive steps, each with increasing demands for scoring acceptable binding positions. The initial step is sufficiently crude, so that all candidate binding sites on the tubulin dimer can be scanned for docking. Following this step, candidate binding sites with the greatest predicted binding strength are passed along for more exact placement into each potential binding site. Completion of all computational steps for docking yields the minimum energy position for each test molecule into its most favorable binding location. 2The molecular coordinates for all binding conformations described in this paper are available on request to D. G. Covell at [email protected]. Direct Photoaffinity Labeling of Tubulin by [3H]Dolastatin 10 and Identification of the Predominant Radiolabeled Peptide—We decided to explore the potential of the direct photoaffinity labeling technique for providing information about the dolastatin 10-binding site on tubulin based on the successful studies with colchicine (17Wolff J. Knipling L. Cahnmann H.J. Palumbo G. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2820-2824Crossref PubMed Scopus (66) Google Scholar), paclitaxel (40Rao S. Horwitz S.B. Ringel I. J. Natl. Cancer Inst. 1992; 84: 785-788Crossref PubMed Scopus (154) Google Scholar), and GTP (9Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar). Only in the latter case was a specific amino acid residue, Cys-12 of β-tubulin, identified, and Shivanna et al. (9Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar) proposed that exposure of tubulin to ultraviolet radiation resulted in generation of sulfhydryl free radicals as the reactive species. Table I summarizes our initial experiments, in which we found that about 8% of the bound [3H]dolastatin 10 reacted covalently with tubulin, and this covalent reaction, as well as the total binding reaction, was substantially inhibited by preincubating the tubulin with a 5-fold molar excess of two highly active analogs of dolastatin 10, auristatin-PE (24Pettit G.R. Srirangam J.K. Barkoczy J. Williams M.D. Boyd M.R. Hamel E. Pettit R.K. Hogan F. Bai R. Chapuis J.-C. McAllister S.C. Schmidt J.M. Anti-Cancer Drug Des. 1998; 13: 243-277PubMed Google Scholar, 25Natsume T. Watanabe J. Tamoki S. Fujio N. Miyasaka K. Kobayashi M. Jpn. J. Cancer Res. 2000; 91: 737-747Crossref PubMed Scopus (50) Google Scholar) and (19aR)-isodolastatin 10 (12Bai R. Roach M.C. Srirangam J.K. Barkoczy J. Pettit G.R. Ludueña R.F. Hamel E. Biochem. Pharmacol. 1993; 45: 1503-1515Crossref PubMed Scopus (81) Google Scholar, 41Bai R. Pettit G.R. Hamel E. Biochem. Pharmacol. 1990; 40: 1859-1864Crossref PubMed Scopus (67) Google Scholar). The cross-linking reaction therefore appeared to be largely specific and to require initial binding of the peptide to the protein.Table IInhibition of total and covalent binding of dolastatin 10 to tubulin by dolastatin 10 analogs Each 0.65-ml reaction mixture contained 10 μm tubulin, 0.1 m Mes (pH 6.9), 0.5 mm MgCl2, and, if present, 50 μm auristatin-PE or 50 μm (19aR)-isodolastatin 10. The mixtures were incubated for 5 min at 22 °C, and 10 μm [3H]dolastatin 10 was added. After an additional 15 min at 22 °C, the reaction mixtures were exposed to UV light on ice as described in the text for 5 min. A 0.2-ml aliquot of each reaction mixture was mixed with 0.3 ml of 8 m guanidine HCl. Triplicate 0.15-ml aliquots from the original reaction mixture and from the aliquot mixed with the denaturant were applied to syringe columns prepared in 0.1 m Mes, 0.5 mm MgCl2 or in 4 m guanidine HCl, as appropriate. Averages obtained from three independent experiments are shown. In these experiments the average total binding of [3H]dolastatin 10 was 0.52 mol/mol tubulin and of covalently bound [3H]dolastatin was 0.040 mol/mol tubulin.Peptide addedTotal dolastatin 10 boundDolastatin 10 cross-linked to tubulin% inhibition ± S.D.Auristatin-PE81 ± 463 ± 4(19aR)-Isodolastatin 1070 ± 1058 ± 6 Open table in a new tab In the experiments summarized in Table I and elsewhere in this study, the stoichiometry of cross-linking ranged from 0.040 to 0.054 mol of dolastatin 10/mol of tubulin. Whereas this stoichiometry is relatively low compared with the results obtained by direct photoaffinity labeling with exchangeable site GDP or GTP (Refs. 9Shivanna B.D. Mejillano M.R. Williams T.D. Himes R.H. J. Biol. Chem. 1993; 268: 127-132Abstract Full Text PDF PubMed Google Scholar and 42Bai R. Choe K. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. 1998; 273: 9894-9897Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar; also see below), it differs little from results obtained with other antimitotic drugs. Direct photoaffinity labeling experiments with [3H]paclitaxel 3S. B. Horwitz, personal communication. (40Rao S. Horwitz S.B. Ringel I. J. Natl. Cancer Inst. 1992; 84: 785-788Crossref PubMed Scopus (154) Google Scholar) and with [3H]colchicine 4J. Wolff, personal communication. and [3H]vinblastine 4J. Wolff, personal communication. (17Wolff J. Knipling L. Cahnmann H.J. Palumb
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