Structural comparison of dimeric Eg5, Neurospora kinesin (Nkin) and Ncd head-Nkin neck chimera with conventional kinesin.

Cryo-electron microscopy and 3D image reconstruction of microtubules saturated with kinesin dimers has shown one head bound to tubulin, the other free. The free head of rat kinesin sits on the top right of the bound head (with the microtubule oriented plus-end upwards) in the presence of 5'-adenylylimido-diphosphate (AMPPNP) and on the top left in nucleotide-free solutions. To understand the relevance of this movement, we investigated other dimeric plus-end-directed motors: Neurospora kinesin (Nkin); Eg5, a slow non-processive kinesin; and a chimera of Ncd heads attached to Nkin necks. In the AMPPNP (ATP-like) state, all dimers have the free head to the top right. In the absence of nucleotide, the free head of an Nkin dimer appears to occupy alternative positions to either side of the bound head. Despite having the Nkin neck, the free head of the chimera was only seen to the top right of the bound head. Eg5 also has the free head mostly to the top right. We suggest that processive movement may require kinesins to move their heads in alternative ways.

Importance of a flexible hinge near the motor domain in kinesin-driven motility.

Conventional kinesin is a molecular motor consisting of an N-terminal catalytic motor domain, an extended stalk and a small globular C-terminus. Whereas the structure and function of the catalytic motor domain has been investigated, little is known about the function of domains outside the globular head. A short coiled-coil region adjacent to the motor domain, termed the neck, is known to be important for dimerization and may be required for kinesin processivity. We now provide evidence that a helix-disrupting hinge region (hinge 1) that separates the neck from the first extended coiled-coil of the stalk plays an essential role in basic motor activity. A fast fungal kinesin from Syncephalastrum racemosum was used for these studies. Deletion, substitution by a coiled-coil and truncation of the hinge 1 region all reduce motor speed and uncouple ATP turnover from gliding velocity. Insertion of hinge 1 regions from two conventional kinesins, Nkin and DmKHC, fully restores motor activity, whereas insertion of putative flexible linkers of other proteins does not, suggesting that hinge 1 regions of conventional kinesins can functionally replace each other. We suggest that this region is essential for kinesin movement in its promotion of chemo-mechanical coupling of the two heads and therefore the functional motor domain should be redefined to include not only the catalytic head but also the adjacent neck and hinge 1 domains.

Reversal in the direction of movement of a molecular motor.

Kinesin and non-claret disjunctional (ncd) are molecular motors of the kinesin superfamily that move in opposite directions along microtubules. The molecular basis underlying the direction of movement is unclear, although it is thought to be an intrinsic property of the motor domain, a conserved region about 330 amino acids in length. The motor domain is found at the amino terminus in conventional kinesins, but at the carboxy terminus in ncd. Here we report on a chimaera composed of the motor domain of the minus-end-directed kinesin of Neurospora crassa. The bacterially expressed fusion protein was tested in motility assays using polarity-marked microtubules. Surprisingly, the chimaera moved towards the plus end, demonstrating that the polarity of force generation of the ncd motor domain has been reversed. This finding indicates that the domain organization, particularly the position of the motor domain, is of fundamental importance for the polarity of force production. It also demonstrates that the direction of microtubule movement is not controlled solely by the motor domain.