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.
Kif18A is a member of the kinesin-8 family was identified as a central component for the correct alignment of chromosomes at the spindle equator. Recent in vitro analyses revealed that Kif18A has a unique dual functionality, motility and depolymerase. Previously, BTB-1 was found to be the first small molecule inhibitor of Kif18A. BTB-1 potently inhibits the ATPase activity of Kif18A (IC50=1.69 mm) but not of other tested key mitotic kinesins. BTB-1 blocks the motility of Kif18A in a reversible manner. BTB-1 inhibits Kif18A in an adenosine triphosphate (ATP)-competitive but microtubule-uncompetitive manner and slows down the progression of cells through mitosis. In our previous study, we demonstrated that the conventional kinesin in which functional sites modified with azobenzene derivative exhibit photo-reversible alteration of ATPase activity accompanied by cis-trans photoisomerization of azobenzene. Interestingly, the backbone structure of cis-azobenzene resembles BTB-1. In this study, we designed and synthesized photochromic BTB-1 analogues composed of azobenzene derivatives in order to regulate ATPase and motor activity of Kif18A in the photo reversible manner. 2-nitro-4-chloroazobenzene (NCAB) is one of the photochromic BTB-1 analogue we have synthesized, exhibited photo-reversible inhibition for the ATPase activity of Kif18A. We also examined the photo reversible effect of NCBA for the ATPase kinetics of Kif18A utilizing the FRET between fluorescent ATP analogue Mant-ATP and the Trp residue of Kif18A mutant F329W. Furthermore, we tried to synthesize BTB-1 analogue that have a photo-crosslinkable azido group for the purpose of photoaffinity labeling to identify the BTB-1 binding site on Kif18A.
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