Department of Chemistry, Binghamton University, State University of New York, Binghamton, New York 13902, USA.
Biochemistry. 2010 Apr 6;49(13):2932-42. doi: 10.1021/bi100159p.
Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.
微管的基本组成部分微管蛋白存在于大多数真核细胞中，作为多个基因产物，称为同工型。主要的微管蛋白同工型在结构和药物结合能力方面高度保守。然而，βVI 型微管蛋白在序列、组装特性和功能上与其他同工型显著不同。它是造血组织中主要的β-微管蛋白同工型，形成血小板边缘带的微管。主要微管蛋白同工型βI、βII、βIII 和βIotaV 与抗微管药物的相互作用已被广泛研究，但关于βVI 型微管蛋白的药物结合特性知之甚少。在这项研究中，我们使用来自鸡红细胞的微管蛋白（CeTb）来表征各种秋水仙碱结合位点配体的活性，CeTb 约 95%为βVI 型。秋水仙碱结合被认为是高等真核生物微管的普遍特性；然而，我们无法在任何实验条件下检测到 CeTb 与秋水仙碱的结合。鬼臼毒素和诺考达唑是结构不同的其他秋水仙碱结合位点配体，能够抑制紫杉醇诱导的 CeTb 组装。令人惊讶的是，秋水仙碱异构体阿考洛辛也能抑制 CeTb 组装，并对 CeTb 表现出可测量的中等亲和力（K(a) = 0.18 x 10(5) M(-1) 与牛脑微管蛋白相比为 5.0 x 10(5) M(-1)）。由于阿考洛辛和秋水仙碱在 C 环结构上有所不同，因此还测试了两种 C 环秋水仙碱类似物与 CeTb 的结合。动力学实验表明，硫代秋水仙碱和氯代秋水仙碱与 CeTb 结合，但结合速度非常缓慢且亲和力较低。CeTb 的分子建模确定了与βI、βII 和βIV 相比，在秋水仙碱结合位点 6A 范围内有五个不同的氨基酸残基；βIII-微管蛋白中的三个氨基酸也发生了改变。有趣的是，改变的氨基酸位于秋水仙碱结合位点的 A 环区域附近，而不是 C 环区域。我们提出，结合位点中的氨基酸差异限制了 CeTb 中 A 环结合域，从而干扰了三甲氧基苯基 A 环的定位，并阻止 C 环结合位点相互作用有效地发生。阿考洛辛能够容纳这种改变的结合模式，因为它的环更小，C 环取代基更灵活。CeTb 同种型βVI 的秋水仙碱结合域序列几乎与其人类造血对应物相同。因此，通过分析配体与 CeTb 的相互作用，有可能发现专门针对人造血细胞中微管蛋白的秋水仙碱结合位点配体。
