Cyclostreptin binds covalently to microtubule pores and lumenal taxoid binding sites.
Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to beta-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of beta-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin.
Buey
Rubén M
RM
Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, Madrid 28040, Spain.
Calvo
Enrique
E
Barasoain
Isabel
I
Pineda
Oriol
O
Edler
Michael C
MC
Matesanz
Ruth
R
Cerezo
Gemma
G
Vanderwal
Christopher D
CD
Day
Billy W
BW
Sorensen
Erik J
EJ
López
Juan Antonio
JA
Andreu
José Manuel
JM
Hamel
Ernest
E
Díaz
J Fernando
JF
D000473:Alkanes / Q000378:metabolism; D000595:Amino Acid Sequence; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D002219:Carbamates / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D000077143:Docetaxel; D034261:Epothilones / Q000378:metabolism; D006801:Humans; D020128:Inhibitory Concentration 50; D007783:Lactones / Q000378:metabolism; D013058:Mass Spectrometry; D008870:Microtubules / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000378:metabolism / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011753:Pyrones / Q000378:metabolism; D043823:Taxoids / Q000378:metabolism / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*; D050257:Tubulin Modulators / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D000473:Alkanes; D002219:Carbamates; D034261:Epothilones; C408303:FR 182877; C413450:Flutax 2; D007783:Lactones; D011083:Polycyclic Compounds; D011753:Pyrones; D043823:Taxoids; D014404:Tubulin; D050257:Tubulin Modulators; D000077143:Docetaxel; D013912:Threonine; C093787:epothilone A; D001216:Asparagine; C071229:discodermolide; D017239:Paclitaxel; C093788:epothilone B