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J Cell Biol
1998 Mar 23;1406:1407-16. doi: 10.1083/jcb.140.6.1407.
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Role of the kinesin neck region in processive microtubule-based motility.
Romberg L
,
Pierce DW
,
Vale RD
.
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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.
Figure 3. Run lengths of single, fluorescently labeled kinesin molecules. Run lengths of 150–270 individual GFP–kinesin molecules moving on axonemes were measured from two independent preparations of each construct. Histograms of the data were plotted using bin widths derived from the formula 2.6σn(−1/3) (Scott, 1979), where σ is the standard deviation of the data and n is the number of data points collected. Exponential curves were fit to the data using only runs >0.25 μm (or 0.2 and 0.15 μm for GLY3 and DEL, respectively), as described in Materials and Methods. Run-length values are shown in Table II.
Figure 2. Fluorescent intensity of individual kinesin molecules. The histograms show the fluorescent intensity of kinesin molecules either moving along axonemes or nonspecifically adsorbed onto the slide surface nearby (refer to Materials and Methods for details). Tick marks represent one arbitrary fluorescent unit; fluorescent intensities cannot be directly compared between preparations because of small variations in laser alignment during different assays.
Figure 4. A structural model for how the kinesin dimer might span the eight nanometers between adjacent α/β tubulin binding sites. In this crystal structure of the rat kinesin dimer (Kozielski et al., 1997), the catalytic core domain is colored blue, the nucleotide is colored gray, the β strand region of the neck (β9 and β10; rat aa 321–336) is colored red, and the neck coiled coil (rat aa 337–370) is colored green (note: the rat kinesin aa numbers differ by −2 aa compared to human kinesin in this region). A side view of a microtubule protofilament from cryoelectron microscopy reconstructions (Hoenger et al., 1995) is shown in gray. The microtubule plus end (the direction of travel for kinesin) is at the right. In A, the unaltered crystal structure of the rat kinesin dimer is shown with one head docked onto the microtubule. The approximate orientation of the bound head was defined by having the half of the molecule containing the nucleotide pointing towards the minus end, the “arrowhead tip” pointing towards the plus end (Hoenger and Milligan, 1997; Sosa et al., 1997), and the main microtubule binding loop (L12) in contact with tubulin surface (Sosa et al., 1997; Woehlke et al., 1997). As noted by Kozielski et al. (1997), the neck coiled coil runs perpendicular to the long axis of the protofilament and is located near but not sterically clashing with the microtubule surface. In the crystal structure, the distance between the two heads is insufficient to enable the second head to dock onto the microtubule. It is important to mention that the structure shown here may not exactly correspond to one that occurs normally in the motility cycle, since the geometry of the heads could be partially determined by crystal contacts and since microtubule or nucleotide binding may change the solution conformation. In B, the β strands between aa 327–336 were separated from the catalytic core in the leading head using the program O (T.A. Jones and M. Kjeldgaard), obeying restraints of bond distances and geometries. This generates a sufficiently long linker to enable the leading head to dock to the adjacent tubulin binding site in the identical orientation to the lagging head. Only modest adjustments need to be made to the neck β strands of the lagging head, since they are already extended and pointing towards the microtubule plus end. In this model, the neck coiled coil does not unwind. The nucleotide (ADP) from the crystal structure is shown in both heads in these panels, although it is more likely that the two heads are in different nucleotide states during the motility cycle.
Amos,
The structure of microtubule-motor complexes.
1997, Pubmed
Amos,
The structure of microtubule-motor complexes.
1997,
Pubmed
Arnal,
Three-dimensional structure of functional motor proteins on microtubules.
1996,
Pubmed
Berliner,
Failure of a single-headed kinesin to track parallel to microtubule protofilaments.
1995,
Pubmed
Block,
Fifty ways to love your lever: myosin motors.
1996,
Pubmed
Block,
Bead movement by single kinesin molecules studied with optical tweezers.
1990,
Pubmed
Bloom,
Motor proteins 1: kinesins.
1995,
Pubmed
Case,
The directional preference of kinesin motors is specified by an element outside of the motor catalytic domain.
1997,
Pubmed
Catterall,
Adenosine triphosphatase from rat liver mitochondria. I. Purification, homogeneity, and physical properties.
1971,
Pubmed
Finer,
Single myosin molecule mechanics: piconewton forces and nanometre steps.
1994,
Pubmed
Funatsu,
Imaging of single fluorescent molecules and individual ATP turnovers by single myosin molecules in aqueous solution.
1995,
Pubmed
Gibbons,
A latent adenosine triphosphatase form of dynein 1 from sea urchin sperm flagella.
1979,
Pubmed
,
Echinobase
Hackney,
Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains.
1995,
Pubmed
Hackney,
Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis.
1994,
Pubmed
Harada,
Mechanochemical coupling in actomyosin energy transduction studied by in vitro movement assay.
1990,
Pubmed
Heim,
Improved green fluorescence.
1995,
Pubmed
Henningsen,
Reversal in the direction of movement of a molecular motor.
1997,
Pubmed
Hirose,
Three-dimensional cryoelectron microscopy of dimeric kinesin and ncd motor domains on microtubules.
1996,
Pubmed
Hirose,
Three-dimensional cryoelectron microscopy of 16-protofilament microtubules: structure, polarity, and interaction with motor proteins.
1997,
Pubmed
Hoenger,
Motor domains of kinesin and ncd interact with microtubule protofilaments with the same binding geometry.
1997,
Pubmed
Hoenger,
Three-dimensional structure of a tubulin-motor-protein complex.
1995,
Pubmed
Howard,
Molecular motors: structural adaptations to cellular functions.
1997,
Pubmed
Howard,
Movement of microtubules by single kinesin molecules.
1989,
Pubmed
Huang,
Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization.
1994,
Pubmed
Huang,
Drosophila kinesin motor domain extending to amino acid position 392 is dimeric when expressed in Escherichia coli.
1994,
Pubmed
Hunt,
Kinesin swivels to permit microtubule movement in any direction.
1993,
Pubmed
Inoue,
Movements of truncated kinesin fragments with a short or an artificial flexible neck.
1997,
Pubmed
Jiang,
Monomeric kinesin head domains hydrolyze multiple ATP molecules before release from a microtubule.
1997,
Pubmed
Jiang,
Influence of the kinesin neck domain on dimerization and ATPase kinetics.
1997,
Pubmed
Kozielski,
The crystal structure of dimeric kinesin and implications for microtubule-dependent motility.
1997,
Pubmed
Kull,
Crystal structure of the kinesin motor domain reveals a structural similarity to myosin.
1996,
Pubmed
Ma,
Interacting head mechanism of microtubule-kinesin ATPase.
1997,
Pubmed
Morii,
Identification of kinesin neck region as a stable alpha-helical coiled coil and its thermodynamic characterization.
1997,
Pubmed
Pierce,
Imaging individual green fluorescent proteins.
1997,
Pubmed
Pierce,
Single-molecule fluorescence detection of green fluorescence protein and application to single-protein dynamics.
1999,
Pubmed
Sablin,
Crystal structure of the motor domain of the kinesin-related motor ncd.
1996,
Pubmed
Sosa,
A model for the microtubule-Ncd motor protein complex obtained by cryo-electron microscopy and image analysis.
1997,
Pubmed
Stewart,
Direction of microtubule movement is an intrinsic property of the motor domains of kinesin heavy chain and Drosophila ncd protein.
1993,
Pubmed
Su,
Effect of chain length on the formation and stability of synthetic alpha-helical coiled coils.
1994,
Pubmed
Svoboda,
Direct observation of kinesin stepping by optical trapping interferometry.
1993,
Pubmed
Tripet,
Demonstration of coiled-coil interactions within the kinesin neck region using synthetic peptides. Implications for motor activity.
1997,
Pubmed
Tucker,
Probing the kinesin-microtubule interaction.
1997,
Pubmed
Uyeda,
The neck region of the myosin motor domain acts as a lever arm to generate movement.
1996,
Pubmed
Vale,
Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins.
1992,
Pubmed
Vale,
Switches, latches, and amplifiers: common themes of G proteins and molecular motors.
1996,
Pubmed
Vale,
Direct observation of single kinesin molecules moving along microtubules.
1996,
Pubmed
Vale,
The design plan of kinesin motors.
1997,
Pubmed
Woehlke,
Microtubule interaction site of the kinesin motor.
1997,
Pubmed