The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to β-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 10⁵ M^–1 in the case of unmodified tubulin to 1.4 ± 0.3 × 10⁵ M^–1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of β-tubulin.

中文翻译：

---

扎曼醇化物与微管蛋白的结合表明紫杉烷位点与秋水仙碱和核苷酸位点的交叉对话

海洋天然产物扎曼醇化物及其类似物构成了具有共价作用机理的紫杉类位点微管稳定剂的新化学型。结合扎曼醇化物的微管蛋白具有易于装配的形式的开关激活环（M-loop），因此代表蛋白质的装配激活状态。在这项研究中，我们表征了共价修饰的活化微管蛋白二聚体的生化特性，并确定了扎曼醇化物对微管蛋白缔合的影响以及微管蛋白配体在其他结合位点的结合。赞帕诺胺活化微管蛋白不会影响其纵向低聚，但会改变其横向缔合特性。扎曼醇化物与β-微管蛋白的共价结合会影响秋水仙碱位点，从而导致结合配体的量子产率发生变化，和可交换的核苷酸结合位点，降低了对核苷酸的亲和力。尽管这些全局作用不会改变2-甲氧基-5-（2,3,4-三甲氧基苯基）-2,4,6-环庚三烯-1-酮（MTC）（秋水仙碱位点的可逆结合剂）的结合亲和力，GTP荧光类似物（Mant-GTP）在核苷酸E位点的结合亲和力从12±2×10降低⁵中号^-1以未修饰的微管蛋白的至1.4±0.3×10的情况下⁵中号^-1中的微管蛋白zampanolide加合物中的情况下，指示所述紫杉烷位点和秋水仙素和β微管蛋白的核苷酸位点之间的信号传输。