Identification of the protein binding region of S-trityl-L-cysteine, a new potent inhibitor of the mitotic kinesin Eg5

Hydrogen-deuterium exchange mass spectrometry is used to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors, in particular for S-trityl-L-cysetine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays. Thus, it is shown that S-trityl-L-cysteine and monastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix alpha3-strand beta5 and loop L5-helix alpha2.