Biochemical and Biophysical characterization of curcumin binding to human mitotic kinesin Eg5: Insights into the inhibitory mechanism of curcumin on Eg5.

In this study we have characterized the biochemical and biophysical interactions of curcumin with the mitotic kinesin Eg5 which plays a pivotal role in the separation of centrosomes during cell division. Curcumin bound to the purified Eg5 (Eg5-437H) with a K value of 7.8μM. The temperature dependent binding analysis and evaluation of thermodynamic parameters indicated the involvement of static quenching mechanism in the binding process. Evidences from competition experiment with monastrol indicated that curcumin bound to Eg5 at a novel druggable site. Using Förster resonance energy transfer the distance between curcumin and monastrol binding site from TRP127 on Eg5-437H was found to be 33Å and 17Å respectively. Curcumin inhibited the ATPase activity of Eg5 motor and perturbed the dynamic interactions between Eg5 and microtubules. Results from circular dichroism studies and molecular dynamics simulations suggest that curcumin binding might perturb the Eg5-437H secondary structure which could be the reason behind its inhibitory effects on Eg5. Cell culture studies performed in HeLa cells indicated that curcumin potentiated the mitotic arrest and monopolar spindle formation in synergism with monastrol, indicating that both ligands could bind simultaneously to the same target.