DEMONSTRATION OF COILED-COIL INTERACTIONS WITHIN THE KINESIN NECK REGION USING SYNTHETIC PEPTIDES - IMPLICATIONS FOR MOTOR-ACTIVITY

Kinesin is a dimeric motor protein that can move for several micromete rs along a microtubule without dissociating. The two kinesin motor dom ains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Rece ntly, a similar to 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferri ng dimerization and processive movement. Based upon its amino acid seq uence, the neck is proposed to dimerize through a coiled-coil interact ion. To determine the accuracy of this prediction and to investigate t he possible function of the neck region in motor activity, we have pre pared a series of synthetic peptides corresponding to different region s of the human kinesin neck (residues 316-383) and analyzed each pepti de for its respective secondary structure content and stability. Resul ts of our study show that a peptide containing residues 330-369 displa ys all of the characteristics of a stable, two-stranded alpha-helical coiled-coil. On the other hand, the NH2-terminal segment of the neck ( residues similar to 316-330) has the capacity to adopt a beta-sheet se condary structure. The COOH-terminal residues of the neck region (resi dues 370-383) are not alpha-helical, nor do they contribute significan tly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and t he extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain co nserved, ''non-ideal'' residues located within the hydrophobic core, w hich we show destabilize the coiled-coil interaction. These residues m ay enable a portion of the coiled-coil to unwind during the mechanoche mical cycle, and we present a model in which such a phenomenon plays a n important role in kinesin motility.