Molecular mechanism of colchicine action: induced local unfolding of beta-tubulin.
Colchicine, the classic antimitotic poison, disrupts cell division by preventing proper assembly of microtubules in the mitotic spindle. Colchicine is known to act by binding to tubulin, the heterodimeric subunit of microtubules. How this binding to tubulin changes the structure of the protein and results in polymerization poisoning has not been characterized. The structural locus of spectroscopically detected conformational changes induced by colchicine is unknown. We report here that colchicine induces the unfolding of a small region in the carboxyl-terminal region of beta-tubulin, around Arg-390. This unfolding is detected by proteolysis with trypsin and chymotrypsin. Chymotrypsin cleaves this region after Phe-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 necessary for assembly. Our results suggest that beta-tubulin is exposed on the growing end of the microtubule, which provides a mechanism for coupling GTP hydrolysis to polymerization.
Sackett
D L
DL
Laboratory of Biochemical Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
Varma
J K
JK
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001923:Brain Chemistry; D002918:Chymotrypsin / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D015151:Immunoblotting; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D010446:Peptide Fragments; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D002918:Chymotrypsin; D014357:Trypsin; D020558:GTP Phosphohydrolases; D003078:Colchicine