Data from "Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state"

KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the ‘K-loop’, which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-head–bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.

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  • KLoopFig1C+D_1Acontrols_RL_Vel.xlsx

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  • KLoopFig1E+F_K1+1Anc_RL_Vel.xlsx

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  • KLoopFig1G_ADP dwells.xlsx

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  • KLoopFig2B+C_1AK1L12_RL_Vel.xlsx

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  • KLoopFig3B_PelletingAssay.xlsx

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  • KLoopFig3C_1AvsSwap_Dwells.xlsx

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  • KLoopFig3D_K1vsK11AL12_Dwells.xlsx

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  • KLoopFig4E+F_BRB12_RL_Vel.xlsx

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  • KLoopFig5C+D_P305L_BRB12_RL+Vel.xlsx

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Metadata

Work Title Data from "Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state"
Access
Open Access
Creators
  1. William O. Hancock
  2. Taylor M. Zaniewski
License CC BY 4.0 (Attribution)
Work Type Dataset
Acknowledgments
  1. Funded by National Institutes of Health grant R35GM139568
Publication Date 2023
DOI doi:10.26207/156h-mc90
Related URLs
Deposited November 14, 2022

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Work History

Version 1
published

  • Created
  • Updated
  • Added Creator WILLIAM O HANCOCK
  • Added Creator Taylor Zaniewski
  • Added KLoopFig1C+D_1Acontrols_RL_Vel.xlsx
  • Added KLoopFig1E+F_K1+1Anc_RL_Vel.xlsx
  • Added KLoopFig2E+F_K11AL12_RL_Vel.xlsx
  • Added KLoopFig3C_1AvsSwap_Dwells.xlsx
  • Updated License Show Changes
    License
    • https://creativecommons.org/licenses/by/4.0/
  • Published

Version 2
published

  • Created
  • Updated Work Title, Publisher, Description, and 1 more Show Changes
    Work Title
    • The net charge of the K-loop regulates KIF1A superprocessivity by enhancing microtubule affinity in the one-head-bound state
    • Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-headbound state
    Publisher
    • Elsevier
    Description
    • KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. We determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine rich insertion in loop-12 termed the ‘K-Loop’, which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native loop-12 of KIF1A with that of kinesin-1, resulted in a 6-fold decrease in run length, and adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell times assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly-bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant compared to wild type. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing the motor binding in its vulnerable one-head-bound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that the KIF1A processivity is linearly dependent on the charge of loop-12.
    • KIF1A is an essential neuronal transport motor protein in the kinesin-3 family, known for its superprocessive motility. However, structural features underlying this function are unclear. Here, we determined that superprocessivity of KIF1A dimers originates from a unique structural domain, the lysine-rich insertion in loop-12 termed the ‘K-loop’, which enhances electrostatic interactions between the motor and the microtubule. In 80 mM PIPES buffer, replacing the native KIF1A loop-12 with that of kinesin-1 resulted in a 6-fold decrease in run length, whereas adding additional positive charge to loop-12 enhanced the run length. Interestingly, swapping the KIF1A loop-12 into kinesin-1 did not enhance its run length, consistent with the two motor families using different mechanochemical tuning to achieve persistent transport. To investigate the mechanism by which the KIF1A K-loop enhances processivity, we used microtubule pelleting and single-molecule dwell time assays in ATP and ADP. First, the microtubule affinity was similar in ATP and in ADP, consistent with the motor spending the majority of its cycle in a weakly bound state. Second, the microtubule affinity and single-molecule dwell time in ADP were 6-fold lower in the loop-swap mutant than WT. Thus, the positive charge in loop-12 of KIF1A enhances the run length by stabilizing binding of the motor in its vulnerable one-headbound state. Finally, through a series of mutants with varying positive charge in the K-loop, we found that KIF1A processivity is linearly dependent on the charge of loop-12, further highlighting how loop-12 contributes to the function of this key motor protein.
    Publication Date
    • 2022
    • 2023
  • Updated Acknowledgments Show Changes
    Acknowledgments
    • Funded by National Institutes of Health grant R35GM139568
  • Renamed Creator William O. Hancock Show Changes
    • WILLIAM O HANCOCK
    • William O. Hancock
  • Renamed Creator Taylor M. Zaniewski Show Changes
    • Taylor Zaniewski
    • Taylor M. Zaniewski
  • Updated Publisher Identifier (DOI) Show Changes
    Publisher Identifier (DOI)
    • https://doi.org/10.1016/j.jbc.2022.102818
  • Added KLoopFig1G_ADP dwells.xlsx
  • Added KLoopFig2D+G_OnRates.xlsx
  • Added KLoopFig2B+C_1AK1L12_RL_Vel.xlsx
  • Added KLoopFig3B_PelletingAssay.xlsx
  • Added KLoopFig3D_K1vsK11AL12_Dwells.xlsx
  • Added KLoopFig3E_HalfSiteRelease.xlsx
  • Added KLoopFig4B+C_SuperK_RL_Vel.xlsx
  • Added KLoopFig4D_SuperK_OnRate.xlsx
  • Added KLoopFig4E+F_BRB12_RL_Vel.xlsx
  • Added KLoopFig4G_BRB12_OnRate.xlsx
  • Added KLoopFig5C+D_P305L_BRB12_RL+Vel.xlsx
  • Added KLoopFig5E_P305L_BRB12_OnRate.xlsx
  • Published
  • Updated Work Title, Publisher, Publisher Identifier (DOI), and 1 more Show Changes
    Work Title
    • Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state
    • Data from "Positive charge in the K-loop of the kinesin-3 motor KIF1A regulates superprocessivity by enhancing microtubule affinity in the one-head–bound state"
    Publisher
    • Elsevier
    Publisher Identifier (DOI)
    • https://doi.org/10.1016/j.jbc.2022.102818
    Related URLs
    • https://doi.org/10.1016/j.jbc.2022.102818