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The forward and backward stepping processes of kinesin are gated by ATP binding

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ABSTRACT

The kinesin motor converts the chemical energy from ATP turnover into mechanical work, which produces successive 8-nm steps in the forward and backward direction along a microtubule. A key problem for kinesin mechanochemistry is explaining how ATP turnover is coordinated with mechanical work. We investigated this by measuring the ATP dependent properties of kinesin forward and backward steps using optical trapping nanometry. The results showed that the rate for both forward and backward steps are ATP-dependent, indicating that ATP binding to kinesin triggers both forward and backward steps. This suggests that ATP turnover in kinesin is not rigidly coupled to total mechanical work at high load.

No MeSH data available.


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Molecular models of kinesin stepping. Kinesin moves in a walking manner using its two head domains. (a) A model in which the backward steps is not coupled with ATP binding. ATP binding to the nucleotide-free head that is attached directly to the microtubule coerces a forward-directed mechanical change in the neck domain forcing the partner head to the forward direction. After the detached head has landed, the alternate head releases from the microtubule as a result of an internal strain between the heads completing the forward step. Backward steps occur because load causes the head to briefly detach, but it quickly reattaches at a nearby backward site. (b) A model in which the backward step is coupled with ATP binding. ATP binding to the nucleotide-free head does not make an ATPase-coupled mechanical change. Instead it relieves the inhibition which allows the partner head to search for adjacent binding sites on the microtubule by undergoing a microtubule-activated ADP release of the partner head. The direction of the step is biased to the forward direction by an asymmetric free energy landscape such as a rachet-like structure or an asymmetric steric effect. The originally bound head releases after the detached head attaches. Letters in the circle at the head represent the prospective binding nucleotide: T=ATP, D=ADP, Pi=phosphate.
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f5-4_11: Molecular models of kinesin stepping. Kinesin moves in a walking manner using its two head domains. (a) A model in which the backward steps is not coupled with ATP binding. ATP binding to the nucleotide-free head that is attached directly to the microtubule coerces a forward-directed mechanical change in the neck domain forcing the partner head to the forward direction. After the detached head has landed, the alternate head releases from the microtubule as a result of an internal strain between the heads completing the forward step. Backward steps occur because load causes the head to briefly detach, but it quickly reattaches at a nearby backward site. (b) A model in which the backward step is coupled with ATP binding. ATP binding to the nucleotide-free head does not make an ATPase-coupled mechanical change. Instead it relieves the inhibition which allows the partner head to search for adjacent binding sites on the microtubule by undergoing a microtubule-activated ADP release of the partner head. The direction of the step is biased to the forward direction by an asymmetric free energy landscape such as a rachet-like structure or an asymmetric steric effect. The originally bound head releases after the detached head attaches. Letters in the circle at the head represent the prospective binding nucleotide: T=ATP, D=ADP, Pi=phosphate.

Mentions: Recent studies support that kinesin moves in a walking manner using alternate microtubule-binding heads18–21. One interpretation is that ATP binding to the nucleotide-free, microtubule-bound head relieves stress allowing the unbound head with ADP bound to land on either of the adjacent microtubule binding sites18,22 (Fig. 5b). The landing direction of the unbound head is biased to the forward direction, possibly by the effect of one or a combination of the neck linker docking23, a strain-based gating mechanism24 or an asymmetric steric effect14. The search by the head to the binding site is powered by the energy from Brownian motion, which causes loose coupling between the ATPase reaction and the total mechanical work done by kinesin. The molecular mechanism behind this may be explained by several physical models including the Feynman’s ratchet model25, the flashing ratchet model26, or the classical Huxley scheme27, where, along with coupling to the mechanical movment, chemical reactions induce non-directional fluctuations or dramatic changes in the energy landscapes. Consequently, the backward steps are not caused by a simple slippage along the microtubule (Fig. 5a) but are coupled to the ATPase reaction. This will be inconsistent with the entire tight mechanochemical coupling mechanism in F1-ATPase28, in which the process of backward steps couple to the ATP synthesis reaction. Futhermore, our results indicate that the detachment of kinesin from the microtubule is an ATP-dependent process. We imagine that most observed detachments were caused by an ATP-bound, attached head undergoing phosphate release before the unbound head attached to a microtubule binding site. The phosphate release caused the attached head to change from strong to weak binding29,30, which in turn would facilitate detachment.


The forward and backward stepping processes of kinesin are gated by ATP binding
Molecular models of kinesin stepping. Kinesin moves in a walking manner using its two head domains. (a) A model in which the backward steps is not coupled with ATP binding. ATP binding to the nucleotide-free head that is attached directly to the microtubule coerces a forward-directed mechanical change in the neck domain forcing the partner head to the forward direction. After the detached head has landed, the alternate head releases from the microtubule as a result of an internal strain between the heads completing the forward step. Backward steps occur because load causes the head to briefly detach, but it quickly reattaches at a nearby backward site. (b) A model in which the backward step is coupled with ATP binding. ATP binding to the nucleotide-free head does not make an ATPase-coupled mechanical change. Instead it relieves the inhibition which allows the partner head to search for adjacent binding sites on the microtubule by undergoing a microtubule-activated ADP release of the partner head. The direction of the step is biased to the forward direction by an asymmetric free energy landscape such as a rachet-like structure or an asymmetric steric effect. The originally bound head releases after the detached head attaches. Letters in the circle at the head represent the prospective binding nucleotide: T=ATP, D=ADP, Pi=phosphate.
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f5-4_11: Molecular models of kinesin stepping. Kinesin moves in a walking manner using its two head domains. (a) A model in which the backward steps is not coupled with ATP binding. ATP binding to the nucleotide-free head that is attached directly to the microtubule coerces a forward-directed mechanical change in the neck domain forcing the partner head to the forward direction. After the detached head has landed, the alternate head releases from the microtubule as a result of an internal strain between the heads completing the forward step. Backward steps occur because load causes the head to briefly detach, but it quickly reattaches at a nearby backward site. (b) A model in which the backward step is coupled with ATP binding. ATP binding to the nucleotide-free head does not make an ATPase-coupled mechanical change. Instead it relieves the inhibition which allows the partner head to search for adjacent binding sites on the microtubule by undergoing a microtubule-activated ADP release of the partner head. The direction of the step is biased to the forward direction by an asymmetric free energy landscape such as a rachet-like structure or an asymmetric steric effect. The originally bound head releases after the detached head attaches. Letters in the circle at the head represent the prospective binding nucleotide: T=ATP, D=ADP, Pi=phosphate.
Mentions: Recent studies support that kinesin moves in a walking manner using alternate microtubule-binding heads18–21. One interpretation is that ATP binding to the nucleotide-free, microtubule-bound head relieves stress allowing the unbound head with ADP bound to land on either of the adjacent microtubule binding sites18,22 (Fig. 5b). The landing direction of the unbound head is biased to the forward direction, possibly by the effect of one or a combination of the neck linker docking23, a strain-based gating mechanism24 or an asymmetric steric effect14. The search by the head to the binding site is powered by the energy from Brownian motion, which causes loose coupling between the ATPase reaction and the total mechanical work done by kinesin. The molecular mechanism behind this may be explained by several physical models including the Feynman’s ratchet model25, the flashing ratchet model26, or the classical Huxley scheme27, where, along with coupling to the mechanical movment, chemical reactions induce non-directional fluctuations or dramatic changes in the energy landscapes. Consequently, the backward steps are not caused by a simple slippage along the microtubule (Fig. 5a) but are coupled to the ATPase reaction. This will be inconsistent with the entire tight mechanochemical coupling mechanism in F1-ATPase28, in which the process of backward steps couple to the ATP synthesis reaction. Futhermore, our results indicate that the detachment of kinesin from the microtubule is an ATP-dependent process. We imagine that most observed detachments were caused by an ATP-bound, attached head undergoing phosphate release before the unbound head attached to a microtubule binding site. The phosphate release caused the attached head to change from strong to weak binding29,30, which in turn would facilitate detachment.

View Article: PubMed Central - PubMed

ABSTRACT

The kinesin motor converts the chemical energy from ATP turnover into mechanical work, which produces successive 8-nm steps in the forward and backward direction along a microtubule. A key problem for kinesin mechanochemistry is explaining how ATP turnover is coordinated with mechanical work. We investigated this by measuring the ATP dependent properties of kinesin forward and backward steps using optical trapping nanometry. The results showed that the rate for both forward and backward steps are ATP-dependent, indicating that ATP binding to kinesin triggers both forward and backward steps. This suggests that ATP turnover in kinesin is not rigidly coupled to total mechanical work at high load.

No MeSH data available.


Related in: MedlinePlus