Chaudhuri A R, Seetharamalu P, Schwarz P M, Hausheer F H, Ludueña R F
Department of Biochemistry, University of Texas Health Science Center at San Antonio, TX 78229, USA.
J Mol Biol. 2000 Nov 10;303(5):679-92. doi: 10.1006/jmbi.2000.4156.
Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on alpha and some supporting a binding site on beta. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the alpha-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on alpha-tubulin; these residues are located well away from the alpha/beta interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the alpha/beta interface of tubulin with the B-ring on the alpha-subunit and the A and the C-rings on the beta-subunit.
微管蛋白是微管的主要结构成分，在细胞分裂过程中积极参与有丝分裂纺锤体的形成和染色体的组织。微管蛋白是多种抗有丝分裂药物的主要作用靶点。其中一些药物，如长春花生物碱和紫杉醇，常用于癌症化疗。不幸的是，由于缺乏有用且合适的工具，我们对这些药物在微管蛋白上的结合位点的了解有限。光亲和标记法是目前可用于检测药物在微管蛋白上结合位点的主要技术。然而，这种方法有几个局限性。首先，只能识别部分结合位点，即与光亲和标记反应的残基。其次，微管蛋白存在一些区域，它们不在结合位点，但会受到药物结合的影响；这些区域无法通过光亲和标记法检测到。第三个，也许也是最严重的局限性是，传统方法可以检测到与配体结合无关但在结合位点一定距离内的区域，该距离小于连接到配体上的光反应性部分的长度。关于秋水仙碱在微管蛋白上结合位点的定位存在很多争议，一些报告表明结合位点在α亚基上，而另一些则支持在β亚基上有结合位点。秋水仙碱对微管蛋白构象也有显著影响，但受影响的区域尚未确定。我们在此尝试通过一种新颖的“足迹”方法来解决这些问题，通过该方法可以确定药物结合位点以及受药物诱导的构象变化影响的微管蛋白结构域。在此，我们首次报告秋水仙碱的B环与α亚基的相互作用会影响一个似乎远离其结合位点的微管蛋白结构域。该结构域包括α微管蛋白上第295、305、315和316位的半胱氨酸残基；这些残基远离秋水仙碱似乎结合的α/β界面。这与秋水仙碱对微管蛋白分子的稳定作用相关。此外，我们还发现秋水仙碱的B环在微管蛋白的稳定性中起主要作用，而A环和C环对其影响很小。因此，我们的结果支持一种模型，即秋水仙碱在微管蛋白的α/β界面结合，B环在α亚基上，A环和C环在β亚基上。
