MOLECULAR MECHANISM OF COLCHICINE ACTION - INDUCED LOCAL UNFOLDING OFBETA-TUBULIN

Colchicine, the classic antimitotic poison, disrupts cell division by preventing proper assembly of microtubules in the mitotic spindle. Col chicine is known to act by binding to tubulin, the heterodimeric subun it of microtubules. How this binding to tubulin changes the structure of the protein and results in polymerization poisoning has not been ch aracterized. The structural locus of spectroscopically detected confor mational changes induced by colchicine is unknown. We report here that colchicine induces the unfolding of a small region in the carboxyl-te rminal region of beta-tubulin, around Arg-390. This unfolding isdetect edbyproteolysiswithtrypsinandchymotrypsin. Chymotrypsincleavesthisregi onafterPhe-389, and trypsin cleaves after Lys-392. The unfolded region appears to be the carboxyl end of an amphipathic helix in the absence of colchicine, and we propose that this unfolding prevents contacts n ecessary for assembly. Our results suggest that beta-tubulin is expose d on the growing end of the microtubule, which provides a mechanism fo r coupling GTP hydrolysis to polymerization.