Photocontrol of mitotic kinesin Eg5 facilitated by thiol-reactive photochromic molecules incorporated into the loop L5 functional loop

Kinesin Eg5 is a plus-end-directed microtubule-based motor that is essential for bipolar spindle formation during eukaryotic cell division. Loop L5 of mitotic kinesin Eg5 is a key region determining ATPase activity and motor function. Photochromic molecules undergo reversible isomerization in response to ultraviolet and visible light irradiation. We introduced three kinds of photochromic molecules, 4-phenylazomaleinanil (PAM), 4-(N-(2-iodoacetyl)amino)-4'-(N-(2-(N-(triphenylmethyl)amino)acetyl)amino)azobenzene (IATAB) and 3,3-dimethyl-1-(2-(2-iodoacetoxy)ethyl)-3H-1,2-dihydroindole-2-spiro-2'-(2H)-6'-nitrochromene (IASP) into L5 to control the Eg5 ATPase activity using light irradiation. We prepared five kinesin Eg5 motor domain mutants, E116C, E118C, Y125C, W127C and D130C, which contained a single reactive cysteine residue in loop L5. The ability of S-trityl-l-cysteine (STLC), a specific Eg5 inhibitor, to inhibit E116C, W127C and D130C was significantly reduced. The photochromic molecules were stoichiometrically incorporated into the cysteine residues in L5 of mutants. W127C and D130C modified with IASP exhibited reversible ATPase activity alterations when subjected to light irradiation-induced photoisomerization. The two IASP modified mutants also demonstrated photocontrolled alterations following treatment with STLC. Additionally, the ATPase activity of the mutant D130C modified with PAM could be photocontrolled. Our findings demonstrate that incorporation of photochromic molecules into the key region of loop L5 facilitates the photocontrol of the function of kinesin Eg5.