Abstract
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.
Highlights
Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating
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
The kinesin heavy chains are organized into four domains: (i) a ϳ325-amino acid residue globular motor domain head that contains the ATP and microtubule binding sites, (ii) a ϳ50-amino acid residue region adjacent to the globular motor domain that is sufficient for allowing dimerization of the motor domains [6] and contains a sequence that is predicted to form an ␣-helical coiled coil [6, 7], (iii) a long (ϳ450-amino acid residue) ␣-helical coiledcoil domain, and (iv) a small globular COOH terminus (8 –11)
Summary
Kinesin is a dimeric motor protein that can move for several micrometers along a microtubule without dissociating. Bacterial expression of the first 340 amino acids of the Drosophila kinesin heavy chain (which contains the core NH2-terminal globular motor domain and the first ϳ10 amino acids of the neck) produces a monomeric protein that generates directed motility when many motors are interacting simultaneously with a single microtubule in gliding motility assays [7, 13]. These monomeric kinesins do not exhibit processive movement when assayed as individual motors in a single molecule fluo-
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