Molecular dissection of the inhibitor binding pocket of mitotic kinesin Eg5 reveals mutants that confer resistance to antimitotic agents.

The mitotic kinesin Eg5 plays an essential role in establishing the bipolar spindle. Recently, several antimitotic inhibitors have been shown to share a common binding region on Eg5. Considering the importance of Eg5 as a potential drug target for cancer chemotherapy it is essential to understand the molecular mechanism, by which these agents block Eg5 activity, and to determine the "key residues" crucial for inhibition. Eleven residues in the inhibitor binding pocket were mutated and the effects were monitored by kinetic analysis and mass spectrometry. Mutants R119A, D130A, P131A, I136A, V210A, Y211A and L214A abolish the inhibitory effect of monastrol. Results for W127A and R221A are less striking, but inhibitor constants are still considerably modified compared to wild-type Eg5. Only one residue, Leu214, was found to be essential for inhibition by STLC. W127A, D130A, V210A lead to increased K(i)(app) values, but binding of STLC is still tight. R119A, P131A, Y211A and R221A convert STLC into a classical rather than a tight-binding inhibitor with increased inhibitor constants. These results demonstrate that monastrol and STLC interact with different amino acids within the same binding region, suggesting that this site is highly flexible to accommodate different types of inhibitors. The drug specificity is due to multiple interactions not only with loop L5, but also with residues located in helices alpha2 and alpha3. These results suggest that tumour cells might develop resistance to Eg5 inhibitors, by expressing Eg5 point mutants that retain the enzyme activity, but prevent inhibition, a feature that is observed for certain tubulin inhibitors.