Failure of a single-headed kinesin to track parallel to microtubule protofilaments

Berliner, Elise; Young, Edgar C.; Anderson, Karin; Mahtani, Hansraj K.; Gelles, Jeff

doi:10.1038/373718a0

Cited by 181 publications

(189 citation statements)

References 22 publications

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“…For mutagenesis, we used Drosophila kinesin, for which there is a well-studied recombinant form (14) and which is highly homologous to the kinesins studied previously (8) (Fig. 2 A-D).…”

Section: Resultsmentioning

confidence: 99%

“…Kinesin mutants were designed from an existing expression plasmid for a recombinant truncated derivative of kinesin, which includes the N-terminal 401 aa of Drosophila melanogaster kinesin heavy chain (DmK401), followed by a biotin carboxyl carrier protein (BCCP) and a His 6 tag (gift of J. Gelles, Brandeis University) (14). To create the 2G mutant, we mutated the gene sequences for residues 9 and 12 by using the QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene) with a single oligonucleotide primer containing the desired mutations (5Ј-CGAGAGATTCCCGGCGAG-GACGGCATCAAAGTGG-3Ј).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Kinesin's cover-neck bundle folds forward to generate force

Khalil

Appleyard

Labno³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

136

182

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Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a ␤-sheet with the neck linker to yield the cover-neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-type. Motility data, as well as kinetic analyses, revealed impairment of the force-generating capacity accompanied by a greater load dependence in the mechanochemical cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the Nterminal ''dangling end,'' unlike the neck linker and nucleotidebinding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a constant assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-neck bundle formation. They also suggest a strategy to generate motors with altered mechanical characteristics by targeting the force-generating element.biological motor ͉ force generation ͉ optical trap ͉ power stroke ͉ motor protein T ranslocating motors, such as kinesins and certain myosins, form a distinct class of proteins that ''walk'' along biofilament tracks to perform a wide range of vital cellular processes (1). A fundamental, yet poorly understood aspect of these motors is the energy transduction mechanism that converts the chemical energy of ATP binding, hydrolysis, and product release into mechanical work. Kinesins and myosins appear to have a common nucleotide sensor (2, 3) yet have evolved different energy conversion mechanisms to achieve a variety of motile properties. In myosin, a series of structural changes leading to the rotation of its lever arm have been identified (4), but the details of the force generation have yet to be established.Likewise, the force generation mechanism of kinesin (herein, we mainly consider Kinesin-1) is not known. Until recently, the only mechanical element considered was the neck linker (NL), which connects the N-terminal motor head to the ␣-helical stalk. This Ϸ12-residue segment is disordered and flexible in the absence of ATP and ''docks'' when ATP binds to the motor head (5). Mutations in the NL impair the motility while preserving ATPase activity and microtubule (MT) binding (6, 7). However, a mechanism for its contribution to force generation is not available, and, in lieu of this, affinity-driven zippering of the NL to the motor head has been assumed. Unlike the structurally well-defined lever arm of myosin, the NL is short and flexible when detached. Furthermore, it interacts only weakly with the motor head (8), drawing f...

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“…For mutagenesis, we used Drosophila kinesin, for which there is a well-studied recombinant form (14) and which is highly homologous to the kinesins studied previously (8) (Fig. 2 A-D).…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Kinesin's cover-neck bundle folds forward to generate force

Khalil

Appleyard

Labno³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

136

182

View full text Add to dashboard Cite

show abstract

“…For kinesin, a protein containing the catalytic core plus the neck linker produces motility, albeit at ¢vefold or more reduced velocities (Berliner et al 1995;Stewart et al 1993;Vale et al 1996). These monomeric constructs are also no longer processive in single-molecule motility assays (Berliner et al 1995;Vale et al 1996). Similarly, a truncated Ncd monomer, consisting of the catalytic core plus 13 residues of the helical neck, produces minus-enddirected microtubule gliding with a reduced velocity of motion (Stewart et al 1993).…”

Section: Functional Evidence For a Role Of The Neck In Kinesin Mechanicsmentioning

confidence: 99%

Searching for kinesin's mechanical amplifier

Vale

Case

Sablin

et al. 2000

Phil. Trans. R. Soc. Lond. B

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Kinesin, a microtubule-based motor, and myosin, an actin-based motor, share a similar core structure, indicating that they arose from a common ancestor. However, kinesin lacks the long lever-arm domain that is believed to drive the myosin power stroke. Here, we present evidence that a much smaller region of ca. 10^40 amino acids serves as a mechanical element for kinesin motor proteins. These`neck regions' are class conserved and have distinct structures in plus-end and minus-end-directed kinesin motors. Mutagenesis studies also indicate that the neck regions are involved in coupling ATP hydrolysis and energy into directional motion along the microtubule. We suggest that the kinesin necks drive motion by undergoing a conformational change in which they detach and re-dock onto the catalytic core during the ATPase cycle. Thus, kinesin and myosin have evolved unique mechanical elements that amplify small, nucleotide-dependent conformational changes that occur in their similar catalytic cores.

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“…Bacterial expression of the first 340 amino acids of the Drosophila kinesin heavy chain (which contains the core NH 2 -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). However, these monomeric kinesins do not exhibit processive movement when assayed as individual motors in a single molecule fluo-rescence motility assay (14) or a bead assay (15). A kinesin motor containing the complete motor and neck domains, on the other hand, forms a dimer and also exhibits processive movement (14,16).…”

mentioning

confidence: 99%

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

Tripet

Vale

Hodges

1997

Journal of Biological Chemistry

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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 ϳ50-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 ␣-helical coiled-coil. On the other hand, the NH 2 -terminal segment of the neck (residues ϳ316 -330) has the capacity to adopt a ␤-sheet secondary structure. The COOH-terminal residues of the neck region (residues 370 -383) are not ␣-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 ␣-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 coiledcoil 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.Understanding how motor proteins generate force and movement from the chemical hydrolysis of ATP remains one of the most intriguing problems in biophysics. At present, there are three separate families of motor proteins found within eukaryotic cells: myosins, which move along actin filaments, and kinesins and dyneins, which move along microtubules (1). The motor domains that typify each of these superfamilies exhibit little or no amino acid sequence similarity, and hence it was believed that they had evolved separately and were structurally unrelated. However, the recently determined crystal structure of kinesin revealed an unexpected structural similarity to the core of the myosin motor domain, particularly in the nucleotide binding pocket (2, 3). Hence, myosin and kinesin may share some similarities in how they generate unidirectional movement and force, although the precise mechanistic details remain to be elucidated for both types of motors.Kinesin has proven to be an excellent model system for investigating the mechanism of motility, in part due to the small size of its motor domain (Ͼ2-fold smaller than myosin's). Kinesin purified from tissue sourc...

show abstract

Failure of a single-headed kinesin to track parallel to microtubule protofilaments

Cited by 181 publications

References 22 publications

Kinesin's cover-neck bundle folds forward to generate force

Kinesin's cover-neck bundle folds forward to generate force

Searching for kinesin's mechanical amplifier

Demonstration of Coiled-Coil Interactions within the Kinesin Neck Region Using Synthetic Peptides

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