Linkage between ligand binding and control of tubulin conformation.
The effect of both antimitotic drugs and nucleotide analogues on the magnesium-induced self-association of purified tubulin into 42S double rings has been examined by sedimentation velocity. In the absence of magnesium, all complexes sedimented as the 5.8S species. The binding of colchicine to tubulin led to a small but consistent (-0.1 to -0.2 kcal/mol) enhancement in the self-association of tubulin alpha-beta dimers. In the absence of nucleotide at the exchangeable site, tubulin retained a weak ability (K2 = 7.5 x 10(3) M-1) to self-associate, which was unchanged by the addition of guanosine or GMP. Analogues with altered P-O-P bonds (GMPPCP, GMPPNP) did not support ring formation at the protein concentrations examined, although GMPPCP supported microtubule assembly. When the exchangeable site was occupied by nucleotides altered on the gamma-phosphate (GTP gamma S, GTP gamma F), rings were formed; tubulin-GTP gamma F formed rings to an extent slightly greater than did tubulin-GTP, and tubulin-GTP gamma S to about the same extent as tubulin-GDP. Both of these analogues are inhibitors of microtubule assembly. These results are consistent with a model [Melki, R., Carlier, M.-F., Pantaloni, D., & Timasheff, S. N. (1989) Biochemistry 28, 9143-9152] in which an equilibrium exists between straight (microtubule-forming) and curved (ring-forming) conformations of tubulin. Furthermore, the present results indicate that the "switch" which controls the nature of the final polymeric product via free energy linkages is the occupancy of the gamma-phosphate binding locus of the exchangeable site by a properly coordinated metal-nucleotide complex.
Shearwin
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KE
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254-9110.
Timasheff
S N
SN
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003078:Colchicine / Q000378:metabolism* / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000378:metabolism* / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000378:metabolism / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D013816:Thermodynamics; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism; D014461:Ultracentrifugation
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D014404:Tubulin; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D008274:Magnesium; D003078:Colchicine