Conformational preferences of a synthetic 30mer peptide from the interface between the neck and stalk regions of kinesin.

The conformation of a synthetic peptide, consisting of 30 amino acids spanning the neck and hinge regions of rat brain kinesin, was investigated by NMR spectroscopy. The peptide extends from K357 to D386 and has the sequence KSVIQHLEVELNRWRNGEAVPEDEQISAKD. A total of 82 distance range constraints and 23 dihedral angle constraints could be obtained from NOESY and E.COSY spectra, respectively. These were used to calculate 500 structures by applying the REDAC algorithm of the software package DYANA. The first half of the peptide matched the helical structure of the neck determined from an X-ray crystal structure of kinesin. This part normally dimerizes into a coiled-coil by virtue of a leucine zipper interaction, but it is alpha-helical even in the monomeric state. The second half (not visible in the X-ray structure because of disorder) contains locally defined structure elements (extended chain, helical loop) connected by flexible joints. This is consistent with the "hinge" function postulated for this domain which is important for kinesin's motility and orientation.

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 micrometers along a microtubule without dissociating. The two kinesin motor domains are thought to move processively by operating in a hand-over-hand manner, although the mechanism of such cooperativity is unknown. Recently, a approximately 50-amino acid region adjacent to the globular motor domain (termed the neck) has been shown to be sufficient for conferring dimerization and processive movement. Based upon its amino acid sequence, the neck is proposed to dimerize through a coiled-coil interaction. To determine the accuracy of this prediction and to investigate the possible function of the neck region in motor activity, we have prepared a series of synthetic peptides corresponding to different regions of the human kinesin neck (residues 316-383) and analyzed each peptide for its respective secondary structure content and stability. Results of our study show that a peptide containing residues 330-369 displays 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 approximately 316-330) has the capacity to adopt a beta-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370-383) are not alpha-helical, nor do they contribute significantly to the overall stability of the coiled-coil, suggesting that these residues mark the beginning of a hinge located between the neck and the extended alpha-helical coiled coil stalk domain. Interestingly, the two central heptads of the coiled-coil segment in the neck contain conserved, "non-ideal" residues located within the hydrophobic core, which we show destabilize the coiled-coil interaction. These residues may enable a portion of the coiled-coil to unwind during the mechanochemical cycle, and we present a model in which such a phenomenon plays an important role in kinesin motility.