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J. Cell Biol.,
Volume 140, Number 6, March 23, 1998 1407-1416
*
* Departments of Cellular and Molecular Pharmacology, and Departments of Biochemistry and Biophysics, University of
California, San Francisco, California 94143; and Kinesin is a dimeric motor protein that can
move along a microtubule for 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 processive 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 processivity using a fluorescence microscope that can visualize single kinesin-GFP molecules moving along a microtubule. Our results show that replacing the kinesin
neck coiled coil with a 28-aa residue peptide sequence
that forms a highly stable coiled coil does not greatly
reduce 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 processivity 10-fold, but surprisingly does not abolish it.
We also demonstrate that processivity is increased by
threefold when the neck helix is elongated by seven
residues. These results indicate that structural features
of the neck coiled coil, although not essential for processivity, can tune the efficiency of single molecule motility.
Howard Hughes Medical Institute, San Francisco, CA 94143
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