Kinesin–microtubule binding depends on both nucleotide state and loading direction

Uemura, Shunsuke; Kawaguchi, Kenji; Yajima, Junichiro; Edamatsu, Masaki; Toyoshima, Yoko Y.; Ishiwata, Shin’ichi

doi:10.1073/pnas.092546199

Cited by 132 publications

(185 citation statements)

References 25 publications

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“…Kinesin mutants with polypeptide insertions between the neck linker and coiled-coil region are less processive (16), presumably because strain between the bound heads is reduced. This interpretation is supported by evidence that external load modulates the affinity of kinesin for nucleotide and MT substrates (17).…”

supporting

confidence: 66%

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Guydosh

Block

2006

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

128

156

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The two-headed kinesin motor harnesses the energy of ATP hydrolysis to take 8-nm steps, walking processively along a microtubule, alternately stepping with each of its catalytic heads in a hand-over-hand fashion. Two persistent challenges for models of kinesin motility are to explain how the two heads are coordinated (''gated'') and when the translocation step occurs relative to other events in the mechanochemical reaction cycle. To investigate these questions, we used a precision optical trap to measure the singlemolecule kinetics of kinesin in the presence of substrate analogs beryllium fluoride or adenylyl-imidodiphosphate. We found that normal stepping patterns were interspersed with long pauses induced by analog binding, and that these pauses were interrupted by short-lived backsteps. After a pause, processive stepping could only resume once the kinesin molecule took an obligatory, terminal backstep, exchanging the positions of its front and rear heads, presumably to allow release of the bound analog from the new front head. Preferential release from the front head implies that the kinetics of the two heads are differentially affected when both are bound to the microtubule, presumably by internal strain that is responsible for the gating. Furthermore, we found that ATP binding was required to reinitiate processive stepping after the terminal backstep. Together, our results support stepping models in which ATP binding triggers the mechanical step and the front head is gated by strain.motor coordination ͉ optical tweezers ͉ single-molecule biophysics ͉ gating ͉ processivity C onventional kinesin, the founding member of the Kinesin-1 family, uses energy from ATP hydrolysis to transport cellular cargo, taking 8-nm steps along microtubules (MTs) (1). Kinesin molecules are formed from two identical heavy chains, whose N-terminal regions fold to form catalytic motor domains, or heads, which are joined by ''neck-linker'' regions to a common, coiled-coil stalk. With each step, the two kinesin heads exchange leading and trailing positions as they alternately hydrolyze ATP, generating ''hand-over-hand'' motion (2-7). This motion is processive under moderate loads for up to Ϸ100 steps in succession, a property that enables small numbers of motors to ferry cargo over long distances (8, 9). The mechanism responsible for the remarkable processivity of kinesin remains unresolved. Furthermore, the relative order of the translocation step with respect to other steps in the mechanochemical cycle is not established.A corollary of kinesin processivity is that its two heads do not function independently, but in concert. Put another way, the relative phase between the heads must be synchronized by a gating mechanism that prevents both heads from dissociating simultaneously from the MT. Because of the 2-fold symmetry imparted by the kinesin structure, this mechanism must break symmetry to permit synchronization. The most attractive candidate for such a mechanism is the mechanical strain that develops when the two heads separate an...

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supporting

confidence: 66%

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Guydosh

Block

2006

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

128

156

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“…Saturating ADP (1 mM) weakens the actomyosin bond and decreases the unbinding force to 4.0 Ϯ 0.1 pN and 3.1 Ϯ 0.1 pN for backward and forward loads, respectively. Such small separation of the peaks is due to only 10-fold difference in the lifetimes of the nucleotide-free and ADP-bound states on actin in the absence of load, contrary to 150-fold difference in case of kinesin (22). However, the t test confirms that for both loading directions the two peaks are statistically distinguishable (twotailed t test; P Ͻ 0.05).…”

Section: Resultsmentioning

confidence: 57%

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

Oguchi

Mikhailenko

Ohki

et al. 2008

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

Self Cite

112

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“…Single-molecule imaging of the fluorescently labeled kinesin motor domains at low ATP levels indicates a predominantly two-heads-bound state [27]. However, unbinding force measurements in the presence of ADP indicate a onehead-bound state [43], and recent mechanical data [44••] and kinetic data [45] suggest that, during the waiting state, one head grips the microtubule while the other diffuses. Reconciling these data demands the differentiation of strongly bound force-bearing states and weakly bound electrostatically tethered states of each motor head, and new experimental methods may be required.…”

Section: Kinesin Processivity: Unresolved Questionsmentioning

confidence: 99%

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

Valentine

Gilbert

2007

Current Opinion in Cell Biology

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Conventional kinesin and Eg5 are essential nanoscale motor proteins. Single-molecule and presteady-state kinetic experiments indicate that both motors use similar strategies to generate movement along microtubules, despite having distinctly different in vivo functions. Single molecules of kinesin, a long-distance cargo transporter, are highly processive, binding the microtubule and taking 100 or more sequential steps at velocities of up to 700 nm/s before dissociating, whereas Eg5, a motor active in mitotic spindle assembly, is also processive, but takes fewer steps at a slower rate. By dissecting the structural, biochemical and mechanical features of these proteins, we hope to learn how kinesin and Eg5 are optimized for their specific biological tasks, while gaining insight into how biochemical energy is converted into mechanical work.

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Kinesin–microtubule binding depends on both nucleotide state and loading direction

Cited by 132 publications

References 25 publications

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain

Load-dependent ADP binding to myosins V and VI: Implications for subunit coordination and function

To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

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