Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation.
Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation.
复制标题
ADP 和 ATP 存在下 monastrol 与 Eg5 变构相互作用的差异：差异 FT-IR 研究。
DOI：
10.1021/bi048982y
复制
发表时间：
2004
期刊：
影响因子：
2.9
通讯作者：
Kim,Sunyoung
中科院分区：
文献类型：
--
作者：
Wojcik,EdwardJ;Dalrymple,NadineA;Alford,ShannonR;Walker,RichardA;Kim,Sunyoung
Eg 5是高等真核生物中有丝分裂进程所需的驱动蛋白样马达蛋白。它被认为是交联反平行微管，并提供形成双极纺锤体所需的力。Monastrol通过Eg 5的变构抑制导致有丝分裂纺锤体的灾难性崩溃。利用截短的Eg 5蛋白，我们采用差分红外光谱探测在ADP或ATP的存在下与monastrol的马达蛋白中发生的结构变化。Eg 5 −monastrol−核苷酸复合物的差示FT-IR光谱表明，在与核苷酸相互作用时，存在对应于马达中二级结构元件的相互转换的触发构象变化。值得注意的是，在ADP存在下引起的构象变化不同于在ATP存在下引起的构象变化。在Eg 5 −monastrol复合物中，ADP的交换与随机结构的减少和α-螺旋含量的增加有关。相反，Eg 5 −monastrol−ATP复合物的形成与α-螺旋含量的减少和伴随的β-折叠含量的增加有关。当ATP而不是ADP在monastrol存在下与马达结构域相互作用时，Eg 5中的一个或多个羧酸残基发生独特的变化。使用这些方法首次直接剖析了抑制剂-蛋白质相互作用，证明了在ADP与ATP存在下monastrol的结构结果存在明显差异。
Eg5 is a kinesin-like motor protein required for mitotic progression in higher eukaryotes. It is thought to cross-link antiparallel microtubules, and provides a force required for the formation of a bipolar spindle. Monastrol causes the catastrophic collapse of the mitotic spindle through the allosteric inhibition of Eg5. Utilizing a truncated Eg5 protein, we employ difference infrared spectroscopy to probe structural changes that occur in the motor protein with monastrol in the presence of either ADP or ATP. Difference FT-IR spectra of Eg5−monastrol−nucleotide complexes demonstrate that there are triggered conformational changes corresponding to an interconversion of secondary structural elements in the motor upon interaction with nucleotides. Notably, conformational changes elicited in the presence of ADP are different from those in the presence of ATP. In Eg5−monastrol complexes, exchange of ADP is associated with a decrease in random structure and an increase in α-helical content. In contrast, formation of the Eg5−monastrol−ATP complex is associated with a decrease in α-helical content and a concomitant increase in β-sheet content. One or more carboxylic acid residues in Eg5 undergo unique changes when ATP, but not ADP, interacts with the motor domain in the presence of monastrol. This first direct dissection of inhibitor−protein interactions, using these methods, demonstrates a clear disparity in the structural consequences of monastrol in the presence of ADP versus ATP.