Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

Abstract

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. 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 the of to the colchicine ring to the or the R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R.F. PubMed Scopus Google Scholar). A number of structurally and with the binding of to tubulin. near of of these drugs the are by the of have that the bind near the with binding to and have that this of tubulin be the R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This of tubulin to be to the exchangeable site on β-tubulin, on two a sulfhydryl cross-link be formed in between (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar, R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar, R.F. PubMed Scopus Google Scholar). In of tubulin by exchangeable site nucleotide to of J. Biol. Chem. Full Text PDF PubMed Google and of R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). drugs that have been inhibit the formation of the with vinblastine the (2Ludueña R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar, M.C. R.F. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Roach M.C. J. R.F. Hamel E. Pharmacol. PubMed Scopus Google Scholar). that inhibit binding to tubulin also inhibit nucleotide on β-tubulin R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Hamel E. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, R. Hamel E. PubMed Scopus Google Scholar, R. Hamel E. Scopus Google Scholar). only nucleotide Hamel E. PubMed Scopus Google Scholar), and R. Hamel E. J. Biol. Chem. 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The dolastatin 10 was in of acid with of A of in acid in was and the was for at was and the was through The by The was on a was with 10 in at 1 The was at The the with a of was to and of was The with that of dolastatin 10, of a at to the atom at to a at on the thiazole The 10 was in of of and of on The was exposed to at for at The was by and the was three with The was on a with The dolastatin 10 was from the with The was by and the was in of The was and to that of dolastatin 10 by and was and the was The a at that the was at in the thiazole for at in exchangeable of analog of with a atom at in the ring in J. Hamel E. 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Chem. 1998; Full Text Full Text PDF PubMed Scopus Google also it from with antimitotic drugs. with S. J. PubMed Scopus Google and with and J. S. A. 1991; PubMed Scopus Google of than of of tubulin. to the tubulin subunit cross-linked to dolastatin 10 it to the tubulin subunits by or by on R. Nguyen N.Y. Hamel E. PubMed Scopus Google Scholar). formation the on the The tubulin was by a with the of protein in the β-tubulin not tubulin that been in the presence of 10 was to acid a from a with A the and the of the The primary site for acid an PubMed Scopus Google Scholar). are only three such sites in tubulin. in the E. S. A. PubMed Scopus Google and two in the and positions E. R. S. A. PubMed Scopus Google Scholar). In studies with β-tubulin cross-linked to colchicine (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. 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Full Text PDF PubMed Google that ultraviolet sulfhydryl residues that a between dolastatin 10 and β-tubulin could be between and the thiazole ring of the with formation of a and of the thiazole protein studies in to the amino acid cross-linked to the to the residues in β-tubulin are at positions and E. R. S. A. PubMed Scopus Google this not at with and the and to a peptide that could be This could be to the of between dolastatin 10 and β-tubulin to the of the amino acid Indirect the between 10 and at J. Biol. Chem. Full Text PDF PubMed Google and R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that of exchangeable site nucleotide at dolastatin 10 potently nucleotide on β-tubulin R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the conformation of dolastatin 10 into the most binding on the electron crystallographic model of tubulin (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar), the sulfur atom of and the thiazole ring of dolastatin 10 in proximity These that the for cross-link formation between and dolastatin 10. A was that could not form a cross-link to β-tubulin. lacking the thiazole ring, this analog of dolastatin 10 has with the in and in the two have been J. Hamel E. R. 1998; Google Scholar, J. S. J. 2000; PubMed Scopus Google Scholar, J. R. Hamel E. Google Scholar, S. Chem. PubMed Scopus Google Scholar). that bound to tubulin 10, but or to the protein to tubulin but to form a cross-link 10 and 10 10 or 10 a at to in the been in to The was for 15 on of was to in the been in and protein in and the are from three in a In an a to that that cross-link formation between bound in the exchangeable site by two of with S. Hamel E. J. PubMed Scopus Google and β-tubulin occurred tubulin at the was R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). the amino acid that formed the with dolastatin 10, of tubulin to form the cross-link J. Biol. Chem. Full Text PDF PubMed Google inhibit cross-link formation with dolastatin 10. This to be the and of tubulin also the of dolastatin 10 to bind to tubulin about of the of dolastatin 10 bound to tubulin was by The of a to formation of the dolastatin cross-link a for the of the this was in the of the drug not the of dolastatin 10 in that binding of dolastatin 10 to tubulin was a for cross-link an on the binding of to tubulin would be the in the exchangeable site been cross-linked to β-tubulin. In contrast, of tubulin a on the binding of or to tubulin The was also in dolastatin 10 inhibited formation between and β-tubulin the tubulin with the in the exchangeable site S. Hamel E. J. PubMed Scopus Google Scholar). also the of a number of drugs that inhibit nucleotide on formation between tubulin and the exchangeable site the only the dolastatin 10 10 and a peptide antimitotic that inhibit the binding of 10 to tubulin cryptophycin or than dolastatin 10 and a with A are by R. In was an of formation by A and cryptophycin well by and most for the of cryptophycin and that of from the exchangeable site the tubulin was to of 10 This between dolastatin 10 and the three antimitotic was inhibit nucleotide on β-tubulin and bind to the protein indicated by the of R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Hamel E. Scopus Google Scholar, R. Hamel E. 1996; Google Scholar). a result in between the and β-tubulin than dolastatin 10 to 10 and inhibit of β-tubulin by bound in the exchangeable site tubulin in the exchangeable site at 10 and the indicated at 15 on the at for of of to each to in the been in from two are in the In the of cross-link formation to the was of cross-link between and 10 in a In a between and the sulfur atom of the thiazole ring of dolastatin 10, from dolastatin 10 from the tubulin. In contrast, J. Biol. Chem. Full Text PDF PubMed Google Scholar, R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), the cross-link formed by between cysteine residues and from by a on of the moiety with formation of not from tubulin. This was in was observed was to the to to the a in the of cross-linked by to but on the of cross-linked to tubulin the between tubulin and dolastatin 10 and formation 10 and 10 or at 10 was at for 15 In the on for in the and was to the for on In the was to the and the on for 1 to the to the end of the of was to each and to in the been in and in the of cross-link formation in the of was tubulin for 10 and for of cross-link in a Modeling electron crystallographic model of tubulin from of antiparallel protofilaments (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google was in this information about the that the binding for dolastatin 10 and of tubulin a of the of β-tubulin in the for ligand-binding sites. in this binding sites was to the exchangeable nucleotide site at the + end of the this binding in a with of for in and in The sulfur atom of in In this the bound in the exchangeable site in and in the docked of dolastatin 10 in in the binding For the of from the electron crystallographic in be that it that dolastatin 10 and paclitaxel bind to the Dolastatin 10 has for tubulin and ring and paclitaxel has for composed of protofilaments, such and The of β-tubulin that was on the to be for E. Downing K.H. Full Text Full Text PDF PubMed Scopus Google Scholar). This in with β-tubulin on the and on the Dolastatin 10 in in docked in this binding site + end binding site from this and indicated by the and at the of the in a site for could the of by dolastatin 10. the binding for at this binding site are than the at the + end binding site in the binding site from the exchangeable nucleotide site by dolastatin 10 and also from the peptide spanning residues 2–31 to dolastatin 10 be cross-linked by the binding model in was in with a in the of β-tubulin in of the exchangeable site in and of the and of dolastatin 10 in for for for and for the sulfur the atom of and the thiazole ring of dolastatin 10. This binding conformation the of nucleotide by dolastatin 10, for the drug and exchangeable nucleotide are in The of the with the ring also In the of cross-link formation between and exchangeable site be R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and the between the sulfur atom and of In this binding conformation for dolastatin 10, the between the sulfur atom and the thiazole sulfur atom of the drug from the cysteine sulfur to the of the thiazole ring are from Å for to Å for This from the cysteine sulfur atom to the thiazole ring consistent with the of of the amino acid to dolastatin 10 with formation with the exchangeable site GDP. in it be that are and but binding of dolastatin 10 to this binding of β-tubulin. In of these the between the sulfur atom and the thiazole ring about have by 10 of the β-tubulin peptide spanning amino acid residues and it highly that of the sulfur at the cysteine residues of tubulin J. Biol. Chem. Full Text PDF PubMed Google Scholar). the only cysteine in the the site of formation with the the dolastatin 10 analog the thiazole ring, has to form an with that this moiety of dolastatin 10 with through formation of a the sulfur could of the of the thiazole A number of consistent with this of formation to and by dolastatin 10 and of formation of their to β-tubulin. the electron crystallographic model of tubulin from by paclitaxel (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar), that the most binding site for dolastatin 10 was to the exchangeable nucleotide site and to This two biochemical of the dolastatin 10 with of cross-link formation R. Roach M.C. J. R.F. Hamel E. Pharmacol. PubMed Scopus Google by between and (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google Scholar, R.F. Roach M.C. Pharmacol. Ther. 1991; 49: 133-152Crossref PubMed Scopus (195) Google Scholar, R.F. PubMed Scopus Google Scholar, R. Ewell J.B. Nguyen N.Y. Hamel E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google of β-tubulin and of nucleotide without of from the exchangeable site R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar). the binding site near to with The of a relatively on a For such to be bind with to their and be specific in that the binding and the are inhibited by the This two in the the an between amino acid and the atom of the it be that the atom be in with an of the binding observed it a In it be dolastatin 10 a of binding to tubulin R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), studies with derivatives of R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D.G. PubMed Scopus Google of Hamel E. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar), a analog S. Chem. PubMed Scopus Google Scholar), and a analog S. Pharmacol. PubMed Scopus Google Scholar). In only two of these studies was a specific peptide in β-tubulin. and J. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google that a vinblastine analog a peptide residues and S. Pharmacol. PubMed Scopus Google that a analog a peptide residues these with of the peptide residues the model a in with these three peptide in The also dolastatin 10 bound in of the sites in with the also 2–31 and are not in the electron crystallographic model (1Nogales E. Wolf S.G. Downing K.H. Nature. 1998; 391: 199-203Crossref PubMed Scopus (1811) Google and not in Dolastatin 10 into the binding site on the in the relatively from the three peptides. In contrast, the dolastatin 10 into the + end binding site to the exchangeable nucleotide site in the of the to of the peptide well the 2–31 peptide but from the have not vinblastine or into the + end binding in A and but of the peptide are near this vinblastine and of vinblastine have binding sites to and and vinblastine are relatively of nucleotide on β-tubulin R. Hamel E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Hamel E. PubMed Scopus Google Scholar). studies with dolastatin 15 and cryptophycin 1 at the + end binding these two antimitotic of the of cryptophycin 1 on the binding of 10 and 15 to tubulin. dolastatin 10 was to bind with to with an of about R. Hamel E. Pharmacol. Google Scholar). 1 the binding of dolastatin 10 to tubulin in a R. Hamel E. 1996; Google Scholar), dolastatin 15 R. Hamel E. Pharmacol. Google Scholar). The of dolastatin 15 to inhibit dolastatin 10 binding from for the of the binding about Z. Hamel E. J. PubMed Scopus Google Scholar). the binding of cryptophycin 1 to tubulin has not been analog cryptophycin to tubulin with an of 2000; PubMed Scopus Google Scholar). the of dolastatin 10 and cryptophycin 1 on the binding of dolastatin 15 to tubulin Z. Hamel E. J. PubMed Scopus Google Scholar), Dolastatin 10 inhibited dolastatin 15 dolastatin 15 from cryptophycin 1 a was of bound dolastatin 15 from tubulin by cryptophycin and about the of cryptophycin 1 was than that of dolastatin for these of dolastatin 10, dolastatin and cryptophycin 1 on each binding to tubulin that the drugs bind to in the the for the binding of the three drugs to the + end binding would be in with the that been that the favored binding of the three drugs are in consistent with the the most of dolastatin 10 and cryptophycin 1 The in the binding consistent with the observed by cryptophycin 1 of dolastatin 10 binding to tubulin. the most of dolastatin 10 and dolastatin 15 The of the consistent with the that the binding dolastatin 10 inhibit the of the binding dolastatin 15 with tubulin. the most of cryptophycin 1 and dolastatin 15 relatively between the consistent with the binding of to tubulin by the of cryptophycin 1 to inhibit dolastatin 15 binding also the of PubMed Scopus Google Scholar), in cryptophycin was into the and into the ring that the the model by these placed cryptophycin in the + end binding the was not to the exchangeable site the model in A and locations of cryptophycin 1 and the exchangeable site in the model are in The model by PubMed Scopus Google the cryptophycin in with the peptide spanning residues with specific between the drug and and to the positions of the two binding sites for by the favored binding of cryptophycin 1 the β-tubulin with the in the of and in and and the exchangeable site in The only the two to have in of a ring of the drug with but this of the the by PubMed Scopus Google not from in the by the to docking In have of dolastatin 10 to the β-tubulin peptide spanning residues 2–31 the exposed to Indirect that the cross-link between the sulfur atom of and the thiazole ring of dolastatin 10. Modeling of dolastatin 10 into the electron crystallographic model of tubulin highly consistent with the biochemical The favored binding site in with the exchangeable nucleotide site and with