Disparity in allosteric interactions of monastrol with Eg5 in the presence of ADP and ATP: a difference FT-IR investigation.

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 alpha-helical content. In contrast, formation of the Eg5-monastrol-ATP complex is associated with a decrease in alpha-helical content and a concomitant increase in beta-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.