Kinesin-14: the roots of reversal
© Cross; licensee BioMed Central Ltd. 2010
Received: 14 July 2010
Accepted: 4 August 2010
Published: 16 August 2010
Kinesin-14 motor proteins step towards microtubule minus ends, in the opposite direction to other kinesins. Work on the still-enigmatic kinesin-14 mechanism published in BMC Structural Biology shows that the carboxyl terminus of the motor head undergoes a dock-undock cycle, like that of plus-end-directed kinesins.
See research article: http://0-www.biomedcentral.com.brum.beds.ac.uk/1472-6807/10/19
Kinesins are the railway engines of the cell, hauling molecular cargo over long distances along microtubule tracks. The kinesin-microtubule railway system is central to the self-organization of eukaryotic life and its mechanisms are consequently an important problem in molecular cell biology. Most kinesins step towards microtubule plus ends, but in one subfamily, the kinesins-14 (K-14), the conventional mechanism is reversed so that the motors haul cargo in the opposite direction, towards microtubule minus ends. Discovering the reversal mechanism is proving challenging. A new crystal structure of a kinesin-14 point mutant  now visualizes for the first time the docking of the proximal part of the kinesin-14 carboxyl terminus to a site on the main part of the kinesin head, showing that the docking and undocking of a carboxy-terminal peptide is a general feature of the mechanism of force generation in both plus-end-directed and minus-end-directed kinesins.
The initial discovery of Ncd [2, 3], the first kinesin-14 to be found, stimulated exciting protein-engineering experiments aimed at finding the structural mechanism for backward stepping. It was quickly established that connecting kinesin-14 heads via their carboxyl termini to the tail of kinesin-1 (kinesin-1 is plus-end-directed) results in a plus-end-directed chimera, whereas connecting kinesin-1 heads via their amino termini to a kinesin-14 tail produces a slow minus-end-directed chimera. So far so good, but subsequent experiments revealed a more subtle and complicated picture. Mutating the head-tail junction in kinesin-14 caused it to revert to plus-end-directed motility , whereas a point mutation near the head-tail junction (Ncd N340K) produces a schizophrenic motor that drives plus-end-directed microtubule sliding for a few seconds, and then switches to driving minus-end-directed sliding, and vice versa . Making sense of these data requires three-dimensional (3D) structural information. Enter crystallography.
Structural studies reveal two stable positions of the coiled-coil tail in kinesin-14
Confidence in some sort of lever-swing model is nonetheless much increased by experiments showing that the ability of Ncd to drive minus-end-directed motility depends critically on the rigidity of the head-proximal part of its coiled-coil tail (often called the 'neck'; the remainder of the tail is called the 'stalk'). Endres et al.  tested the effects of inserting flexible linkers at the head-tail junction, and within the coiled-coil tail, proving firmly that a two-chain coiled tail is a prerequisite for coupling microtubule-activated ATP turnover to microtubule sliding motility. These results explain why wild-type kinesin-14 likes to dimerize, either with itself, as for Ncd, or, as in Kar3, with a non-motor partner protein (Cik1 or Vik1). Dimerization can stiffen the lever, via coiled-coil formation. The experiments of Endres et al.  also confirm that the speed of kinesin-14-driven microtubule sliding increases in direct proportion to the predicted lever length.
Coupling between docking of the kinesin carboxyl terminus and force generation
For the plus-end-directed kinesin-1, kinesin-3 and kinesin-5, crystallography has revealed that the flexible carboxyl terminus of the head (called the 'neck-linker') can dock into a slot on the main part of the head, with the amino terminus lying alongside or wrapping over it. Recent simulations suggest that in kinesin-1, this interaction between the amino and carboxyl termini contributes substantially to the free energy of neck-linker docking , lending new life to the proposal, originally made a decade ago , that neck-linker docking is itself the force-generating event for kinesin-1, equivalent to the lever arm action proposed for myosins. The carboxyl terminus of kinesin-14 is not seen in existing crystal structures. But in the new kinesin-14 crystal structure of Heuston et al. , the carboxyl terminus of one of the two heads is seen to anneal into a site on the main part of the head in much the same way as the carboxy-terminal neck-linker of plus-end-directed kinesins. The docking occurs in the head with the 'post-stroke' lever position, suggesting that lever motion and docking are coupled. Both heads in the new structure contain ADP, but the occupancy is reduced in the head with the docked carboxyl terminus and post-stroke lever position. Heuston et al. speculate that their post-stroke structure may correspond to a no-nucleotide microtubule-bound state of the motor, so that force generation is coupled to ADP release.
Here again, high-resolution cryoelectron microscopy can help. Hirose and colleagues  examined Kar3, a minus-end-directed kinesin-14, and found large-scale melting of the alpha4 relay helix on going from the state with bound ATP analog AMPPNP to the empty state, but any effects on the carboxyl terminus or coiled-coil tail are unknown as they were not included in the motor construct used. Movement of the alpha4 helix and docking of the neck-linker has been confirmed in several plus-end-directed kinesins in complex with microtubules .
Rooting out the reversal mechanism
The new work of Heuston and colleagues  shows that in kinesin-14, as in other members of its extended family, docking and undocking of the carboxy-terminal residues of the head can occur during ATP turnover. The outstanding problem now is to work out the molecular mechanism by which the coiled-coil lever of kinesin-14 is driven from pre-stroke to post-stroke positions and back again. One possibility is that residues on the microtubule contribute. Whatever the answer may be, it is a safe bet that the kinesin-14 mechanism holds yet more surprises in store.
I thank Linda Amos, Andrew McAinsh and Anne Straube for helpful comments and Marie Curie Cancer Care for program support.
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