The net charge of the K-loop regulates KIF1A 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. 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.

Citation

HANCOCK, WILLIAM O; Zaniewski, Taylor (2022). The net charge of the K-loop regulates KIF1A superprocessivity by enhancing microtubule affinity in the one-head-bound state [Data set]. Scholarsphere. https://doi.org/10.26207/156h-mc90

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Work Title The net charge of the K-loop regulates KIF1A superprocessivity by enhancing microtubule affinity in the one-head-bound state
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Open Access
Creators
  1. WILLIAM O HANCOCK
  2. Taylor Zaniewski
License CC BY 4.0 (Attribution)
Work Type Dataset
Publication Date 2022
DOI doi:10.26207/156h-mc90
Deposited November 14, 2022

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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
  • Updated

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
  • Updated