title: Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine.
abstract: The colchicine analog 3-chloroacetyl-3-demthylthio-colchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degree C, and covalently reacts with beta-tubulin at 37 degree C, but not at 0 degree 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 A. using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[14C] Carbonyl)-3- demethylthiocolchicine ([14C]3CTC). This beta-tubulin that had reacted with 3-(chloromethyl-[14C]carbonyl)- 3-demethythiocolchicine ([14C]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys-C, and the radio-labeled 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 A 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 A 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.
authors.affiliation: Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: R
authors.initials: R
authors.lastname: Bai
authors.forename: X F
authors.initials: XF
authors.lastname: Pei
authors.forename: O
authors.initials: O
authors.lastname: Boyé
authors.forename: Z
authors.initials: Z
authors.lastname: Getahun
authors.forename: S
authors.initials: S
authors.lastname: Grover
authors.forename: J
authors.initials: J
authors.lastname: Bekisz
authors.forename: N Y
authors.initials: NY
authors.lastname: Nguyen
authors.forename: A
authors.initials: A
authors.lastname: Brossi
authors.forename: E
authors.initials: E
authors.lastname: Hamel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003078:Colchicine / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D003488:Cyanogen Bromide; D003545:Cysteine / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014404:Tubulin; C076932:3-chloroacetyl-3-demethylthiocolchicine; D003545:Cysteine; D003488:Cyanogen Bromide; D003078:Colchicine