| Abstract
| - Human Eg5, a mitotic motor of the kinesin superfamily, is involved in the formation andmaintenance of the mitotic spindle. The recent discovery of small molecules that inhibit HsEg5 by bindingto its catalytic motor domain leading to mitotic arrest has attracted more interest in Eg5 as a potentialanticancer drug target. We have used hydrogen−deuterium exchange mass spectrometry and directedmutagenesis to identify the secondary structure elements that form the binding sites of new Eg5 inhibitors,in particular for S-trityl-l-cysteine, a potent inhibitor of Eg5 activity in vitro and in cell-based assays.The binding of this inhibitor modifies the deuterium incorporation rate of eight peptides that define twoareas within the motor domain: Tyr125−Glu145 and Ile202−Leu227. Replacement of the Tyr125−Glu145region with the equivalent region in the Neurospora crassa conventional kinesin heavy chain preventsthe inhibition of the Eg5 ATPase activity by S-trityl-l-cysteine. We show here that S-trityl-l-cysteine andmonastrol both bind to the same region on Eg5 by induced fit in a pocket formed by helix α3−strand β5and loop L5−helix α2, and both inhibitors trigger similar local conformational changes within the interactionsite. It is likely that S-trityl-l-cysteine and monastrol inhibit HsEg5 by a similar mechanism. The commoninhibitor binding region appears to represent a “hot spot” for HsEg5 that could be exploited for furtherinhibitor screening.
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