Carbon-13 Nuclear Magnetic Resonance Study of Microtubule Protein: Evidence for a Second Colchicine Site Involved in the Inhibition of Microtubule Assembly

A ¹³C nuclear magnetic resonanee study of bovine microtubule protein was carried out at 43 kG in the presence and absence of colchicine ¹³C labeled at the tropolone methoxy. Analysis indicated that tubulin has at least two colchicine binding sites: a quasi-irreversibly bound, high-affinity site (i.e., the KD 5 μM site generally accepted as the site of colchicine action) as well as a low-affinity site(s) (KD ~ 650 μM) with which free colchicine rapidly exchanges (>100 s-1). The methoxy resonanee is broadened to different apparent extents as a result of binding at these two sites (50- vs. 150-Hz broadening for the high- and low-affinity sites, respectively) but undergoes no change in chemical shift upon binding. The low-affinity sites are interpreted to be analogous to the sites deduced by Schmitt and Atlas [Schmitt, H., & Atlas, D. (1976) J. Mol. Biol. 102, 743-758] from labeling studies using bromocolchicine. These sites are likely to be the sites responsible for the abrupt halt in microtubule assembly (“capping”) observed at high colchicine concentrations (>20 μM)a qualitatively different behavior from that observed at low colchicine concentrations [Sternlicht, H., Ringel, I., & Szasz, J. (1983) Biophys. J. 42, 255-267], Carbon-13 spectra from the aliphatic carbons of microtubule protein consist of narrow resonances many with line widths < 30 Hz superimposed on a broad background. The narrow resonances were assigned to flexible regions in nontubulin proteins [mi-crotubule-associated proteins (MAPs)], in accord with an earlier 1H nuclear magnetic resonacee study of microtubule protein [Woody, R. W., Clark, D. C, Roberts, G. C. K., Martin, S. R., & Bayley, P. M. (1983) Biochemistry 22, 2186-2192]. This assignment was supported by C NMR analysis of phosphocellulose-purified (MAP-depleted) tubulin as well as heat-stable MAPs. Aliphatic carbons in the MAP preparations were characterized by naroow resonances indicative of carbons with considerable motional freedom whereas the aliphatic regions of phosphocellulose-purified tubulin were, for the most part, characterized by broad resonances indicative of carbons with restricted mobility. However, a moderately narrow resonance (≲50-Hz line width) coincident with the C7 resonance of glutamate was detected in ¹³C NMR spectra of tubulin which indicated that a fraction of the glutamic acid residues is relatively mobile. These mobile residues are likely to be at the carboxy-terminus ends, regions rich in glutamates, where secondary structure considerations suggest flexibility.