Carbon-13 nuclear magnetic resonance study of microtubule protein: evidence for a second colchicine site involved in the inhibition of microtubule assembly

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Abstract
A 13C NMR study of bovine microtubule protein was carried out at 43 kG [kilogauss] in the presence and absence of colchicine 13C labeled at the tropolone methoxy. Analysis indicated that tubulin has at least 2 colchicine binding sites: a quasi-irreversibly bound, high-affinity site (i.e., the Kd < 5 .mu.M site generally accepted as the site of colchicine action) as well as a low-affinity site(s)(Kd .apprx. 650 .mu.M) with which free colchicine rapidly exchanges (> 100 s-1). The methoxy resonance is broadened to different apparent extents as a result of binding at these 2 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 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 .mu.M).sbd.a qualitatively different behavior from that observed at low colchicine concentrations. Carbon-13 spectra from the aliphatic carbons of microtubule protein consist of narrow resonances.sbd.many with line widths < 30 Hz.sbd.superimposed on a broad background. The narrow resonances were assigned to flexible regions in nontubulin proteins [microtubule-associated proteins (MAP)], in accord with an earlier 1H NMR study of microtubule protein [Wood et al. (1983)]. This assignment was supported by 13C NMR analysis of phosphocellulose-purified (MAP-depleted) tubulin as well as heat-stable MAP. Aliphatic carbons in the MAP preparations were characterized by narrow 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 (.ltorsim. 50-Hz line width) coincident with the C.gamma. 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.