The fast and superprocessive KIF1A predominately resides in a vulnerable one-head-bound state during its chemomechanical cycle

Kinesin-3 are the fastest and most processive motors of the three neuronal transport kinesin families, yet the sequence of states and rates of kinetic transitions that comprise the chemomechanical cycle are poorly understood. We used stopped-flow fluorescence spectroscopy and single-molecule motility assays to delineate the chemomechanical cycle of the kinesin-3, KIF1A. Our bacterially expressed KIF1A construct, dimerized via a kinesin-1 coiled-coil, exhibits fast velocity and superprocessivity behavior similar to wild-type KIF1A. We established that the KIF1A forward step is triggered by hydrolysis of ATP and not by ATP binding, meaning that KIF1A follows the same chemomechanical cycle as established for kinesin-1 and-2. The ATP-triggered half-site release rate of KIF1A was similar to the stepping rate, indicating that during stepping, rear-head detachment is an order of magnitude faster than in kinesin-1 and kinesin-2. Thus, KIF1A spends the majority of its hydrolysis cycle in a one-head-bound state. Both the ADP off-rate and the ATP on-rate at physiological ATP concentration were fast, eliminating these steps as possible rate limiting transitions. Based on the measured run length and the relatively slow off-rate in ADP, we conclude that attachment of the tethered head is the rate limiting transition in the KIF1A stepping cycle. The fast speed, superprocessivity and load sensitivity of KIF1A can be explained by a fast rear head detachment rate, a rate-limiting step of tethered head attachment that follows ATP hydrolysis, and a relatively strong electrostatic interaction with the microtubule in the weakly-bound post-hydrolysis state.

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Work Title The fast and superprocessive KIF1A predominately resides in a vulnerable one-head-bound state during its chemomechanical cycle
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Open Access
Creators Taylor Zaniewski; William O Hancock
Version number 1
Keyword stopped flow; kinetics; kinesin; kinesin-3; microscopy
License Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Work Type Article
DOI doi:10.26207/krqk-xk50
Deposited September 18, 2020 11:36

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Version 1
published

  • Created
  • Added BRB80_KIF1A560GFP_40pM_PNP2mM_5fps.tif
  • Added TIRF_KIF1A_Motility_PaperData.xlsx
  • Added BRB80_KIF1A560GFP_20pM_ATPgS2mM_10fps.tif
  • Added ATPase_KIF1A-WT_PaperData.xlsx
  • Added kon_Mt_KIF1A406wt_PaperData.xlsx
  • Added KIF1A_Nuc-triggered_Half-SiteRelease_PaperData.xlsx
  • Added Unstrained_mADP_Exchange_Rate_KIF1A406wt_PaperData.xlsx
  • Added Final_Cycle_State_Durations_PaperData.xlsx
  • Added BRB80_KIF1A560GFP_40pM_PNP1mM_ATP1mM_5fps.tif
  • Added Half-Site_Reactivity_Paper_Data.xlsx
  • Added BRB80_KIF1A560GFP_50pM_ATP2mM_5fps.tif
  • Added TIRF_KIF1A_Dwell_Times__PaperData.xlsx
  • Added BRB80_KIF1A560GFP_Apo_2fsp.tif
  • Added Creator Taylor Zaniewski
  • Added Creator William O Hancock
  • Published