Photocontrol of Mitotic Kinesin Eg5 by Incorporating of Photochromic Molecule into the Functional Loop L5

Abstract

All member of kinesin superfamily contain a structurally conserved loop L5 near the ATP binding site. The length of the L5 vary among kinesin superfamily members. It is believed that L5 of kinesin is important region for motor function. Interestingly mitotic kinesin Eg5 has the longest L5 in comparing with other kinesins. The kinesin Eg5 is a microtubule plus-end directed homotetrameric molecular motor that is essential for the formation of a bipolar spindle during eukaryotic cell division. It has been demonstrated that L5 of Eg5 performed as a stabilizer for the Eg5-specific inhibitors (STLC, monastrol) complexes. These inhibitors bind to the same pocket on the Eg5 motor domain composed by of loop L5, α2 and α3. In this study, we tried to photo control of Eg5 ATPase activity by incorporating of photochromic molecule into L5. We prepared 5 mutants of Eg5 which have a single cysteine in L5 in order to incorporate thiol-reactive photochromic molecules. We also synthesized thiol-reactive azobenzene derivative, iodoacetyl-trity-azobenzene (IATAB) and spiropyran derivative, iodoacetyl-spiropyran (IASP). Azobenzene is photoisomerized to trans form of hydrophobic by visible light irradiation, and to cis form of hydrophilic by UV light irradiation. Spiropyran is photoisomerized to zwitterionized merocyanine by UV light irradiation. Merocyanine is converted to hydrophobic spiropyran by visible light irradiation. Photochromic molecules were incorporated into the mutants stoichiometrically. The Eg5 mutant W127C and D130C modified with IASP showed reversible alteration of microtubule dependent ATPase activity upon UV and visible light irradiations. In addition, we also succeed in photo control of inhibitory effect of IASP-W127C and IASP-D130C by STLC.