The Mechanism of Determining the Directionality of NCD

Abstract

Kinesins are the unidirectional motor proteins, moving on microtubules and being involved in various cellular functions. A motor domain of kinesin comprises the motor core: the ATPase catalytic site and MT-binding site, and the neck region: adjacent to the motor core, and the N or C-terminal region: protruding from the motor core. It is known that neck region and N or C-terminal regions are essential for kinesin's motor activity. Among the kinesin superfamily, kinesin-1 moves toward the plus-end of the microtubule while Ncd is the minus-end directed motor. This difference of directionality has long been studied. A series of experiments using chimera motors whose components are exchanged between kinesin-1 and Ncd demonstrated that the neck region was the determinant of the directionality. However the chimera kinesin-1, whose neck was exchanged with Ncd, also exchanged its C-terminal region with Ncd. Thus, it is uncertain whether the C-terminal region of Ncd is the determinant of the minus-ended directionality. To test this, we engineered other chimeras. A chimera kinesin-1 having only the neck of Ncd did not change the directionality. And another chimera kinesin-1 having both the neck and the C-terminal region of Ncd changed into the minus-ended motor. These results indicate that not only the neck but also the C-terminal region of Ncd is indispensable to produce the minus-end directed motility. We then investigated whether Ncd motor core itself has directionality. We engineered a mutant Ncd whose neck was replaced with an artificial peptide, which is thought not to interact with the motor core. This mutant showed the plus-ended directionality. We conclude that Ncd motor core itself has ‘default’ plus-end directionality and an appropriate interaction among the neck, the C-terminal region, and the motor core of Ncd makes Ncd the minus-ended motor.