ROLE OF THE KINESIN NECK REGION IN PROCESSIVE MICROTUBULE-BASED MOTILITY

Kinesin is a dimeric motor protein that can move along a microtubule f or several microns without releasing (termed processive movement). The two motor domains of the dimer are thought to move in a coordinated, hand-over-hand manner. A region adjacent to kinesin's motor catalytic domain (the neck) contains a coiled coil that is sufficient for motor dimerization and has been proposed to play an essential role in proces sive movement. Recent models have suggested that the neck enables head -to-head communication by creating a stiff connection between the two motor domains, but also may unwind during the mechanochemical cycle to allow movement to new tubulin binding sites. To test these ideas, we mutated the neck coiled coil in a 560-amino acid (aa) dimeric kinesin construct fused to green fluorescent protein (GFP), and then assayed p rocessivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show tha t replacing the kinesin neck coiled coil with a 28-aa residue peptide sequence that forms a highly stable coiled coil does not greatly reduc e the processivity of the motor. This result argues against models in which extensive unwinding of the coiled coil is essential for movement . Furthermore, we show that deleting the neck coiled coil decreases pr ocessivity 10-fold, but surprisingly does not abolish it. We also demo nstrate that processivity is increased by threefold when the neck heli x is elongated by seven residues. These results indicate that structur al features of the neck coiled coil, although not essential for proces sivity, can tune the efficiency of single molecule motility.