Research ArticleBIOPHYSICS

A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health

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Science Advances  30 Apr 2021:
Vol. 7, no. 18, eabf1002
DOI: 10.1126/sciadv.abf1002
  • Fig. 1 Characterization of KIF1AP305L in a C. elegans model.

    (A) Cartoon schematic showing the experimental approach. Ablation of the C. elegans homolog of KIF1A, unc-104, leads to the uncoordinated phenotype in which mutant worms are unable to move robustly. In this genetic background, introduction of a WT human KIF1A gene partially rescues animal movement. Human KIF1AP305L was introduced and assayed for its ability to rescue animal motility. (B) The velocity of the worm movement is plotted for each genetic background. Three independent lines were assayed for human KIF1A, and two independent lines were assayed for human KIF1AP305L. Note that while human KIF1A rescues worm movement to approximately half of the normal worm velocity, KIF1AP305L is unable to do so. Data points represent individual worms. Statistical differences between conditions were assessed by Kruskal-Wallis test. ns, not significant; ***P ≤ 0.001, ****P ≤ 0.0001.

  • Fig. 2 Visualization of the location of the P305 residue and 310 helix within the kinesin motor domain.

    (A and B) KIF1A [yellow; Protein Data Bank (pdb): 1vfx], with K-loop/L12 (cyan-green; 288-303) modeled via Swiss-Model (55) and shown for comparison to loop L12 in KIF5B (green; pdb: 4hna). */star, β-tubulin C terminus. 310 helix (red); surrounding α helices: α4 and α5. P305 in KIF1A and corresponding P276 in KIF5B are shown as sphere representations (orange). (C) Sequences of motor domains with available structures were aligned using ClustalW (56). Residues surrounding the 310 helix are shown. Dashed line denotes the original designation of L12, although we show here that the PYRD sequence most often folds into a 310 helix and is thus not a “loop” by conventional definition. Annotation (left): gene name, species abbreviation, pdb id. Right: KIF1A magnesium (Mg)–nucleotide–bound form. Species: Hs/Homo sapiens, Rn/Rattus norvegicus, Mm/Mus musculus, Dm/Drosophila melanogaster, Gi/Giardia intestinalis, Nc/Neurospora crassa. Assignment of secondary structure was done using Procheck (57, 58) according to the method of Kabsch and Sander (59). Background coloring: α helix (blue), 310 helix (orange), hydrogen-bonded turn (yellow), bend (green), without assignment (white), and residues not available in pdb (gray).

  • Fig. 3 Characterization of the KIF1AP305L mutation using full-length motors.

    (A) SEC chromatogram showing elution profiles of full-length KIF1A (magenta) and KIF1AP305L (green). Elution positions of prep contaminants (Contam.) and full-length KIF1A are noted. Below: Coomassie-stained SDS–polyacrylamide gel electrophoresis (PAGE) gel of the column elution fractions. (B) Kymograph from TIRF-M movie showing both KIF1A (magenta) and KIF1AP305L (green) motors moving along the same MT. Equal concentration of each motor was added to the TIRF chamber. (C) Measured velocity distribution of full-length KIF1A motors (N = 2 independent trials and n = 280,270 motors from KIF1A and KIF1AP305L, respectively). ****P< 0.001. (D) Cumulative frequency plot of the measured run lengths of WT and P305L motors. Connecting lines show fit to a one-phase exponential decay function (R2 = 0.99 and 0.98 for KIF1A and KIF1AP305L, respectively). The characteristic run length derived from the fits (τ) is displayed (N = two independent trials and n = 207,241 for KIF1A and KIF1AP305L, respectively).

  • Fig. 4 Characterization of the KIF1AP305L mutation using truncated, constitutively active motors.

    (A) SEC chromatograms for WT (magenta) and P305L truncated (green), dimerized motors (1-393-LZ). Elution positions of prep contaminants and truncated KIF1A are noted. Below: Coomassie-stained SDS-PAGE gel of the column elution fractions encompassing the motor peak. (B) Kymograph from a TIRF-M movie showing both KIF1A (magenta) and KIF1AP305L (green) moving along the same MT. Note the difference in motor concentration. (C) Measured velocity distribution of 1-393-LZ KIF1A motors (N = 2 independent trials and n = 161,116 motors from KIF1A and KIF1AP305L, respectively). (D) Cumulative frequency plot of the measured run lengths of WT and P305L 1-393-LZ motors. Connecting lines show fit to a one-phase exponential decay function (R2 = 0.99 for both KIF1A and KIF1AP305L, respectively). The characteristic run length derived from the fits (τ) is displayed (N = 2 independent trials and n = 142,129 for KIF1A and KIF1AP305L, respectively). (E) Measured number of processive motors per unit length of MT and time (N = 2 independent trials and n = 12 MTs analyzed each for KIF1A and KIF1AP305L). For all panels, ***P ≤ 0.001 and ****P ≤ 0.0001.

  • Fig. 5 Characterization of the weak MT-binding state of KIF1A.

    (A) Kymographs from TIRF-M movies showing binding and dwell events of 1-393-LZ motor constructs in 2 mM ADP. Note the different protein concentrations used. (B) Quantification of the landing rate for KIF1A and KIF1AP305L or KIF1AP305W motors (N = 2 independent trials and n = at least 12 MTs analyzed each for each motor). (C) Cumulative frequency plot of the measured dwell times of WT and P305L 1-393-LZ motors. Connecting lines show fit to a one-phase exponential decay function (R2 > 0.99 for all fits). The characteristic dwell time derived from the fits (τ) is displayed (N = 2 independent trials and n = 196, 221, and 270 for KIF1A, KIF1AP305L, and KIF1AP305W, respectively). (D) Steady-state ATPase assays for KIF1A and KIF1AP305L. Plotted are the mean values from at least two to three independent trials per concentration of MTs, and error bars are SEM. Nonlinear fits to the Michaelis-Menten equation are shown, and the calculated Km and Vmax are displayed in the table to the right with 95% CI errors shown. Note that the errors of the fit for KIF1AP305L cannot be accurately calculated by this method (ud, undetermined).

  • Fig. 6 Characterization of conserved proline mutation in KIF5.

    (A) Identically scaled TIRF-M images showing MTs (blue) and KIF5 molecules (magenta) in ATP at two different protein concentrations. Note the lack of MT binding for KIF5P276L. KIF5BP276L binds robustly to MTs in the presence of AMP-PNP, demonstrating that the motor is still proficient at binding. (B) Kymographs from TIRF-M movies of KIF5B motors in ATP demonstrate processive movement along MTs (diagonal lines) for KIF5B, but not for KIF5BP276L. Only transient binding events are observed for KIF5BP276L. (C) Quantification of mean fluorescence intensity of KIF5B motors along MTs in the presence of ATP at two different protein concentrations (N = 2 independent trials and n = 20 to 35 MTs quantified per condition). a.u., arbitrary units.

  • Fig. 7 Characterization of the force generation properties of KIF1AP305L and KIF5BP276L.

    (A to D) Representative force versus time records of bead movement driven by single molecules of (A) WT KIF1A (1-393)-LZ, (B) KIF1AP305L (1-393)-LZ, (C) WT KIF5B (1-420), and (D) KIF5BP276 (1-420). (E) Detachment forces. Green bars indicate the median values with quartiles. WT KIF1A (1-393)-LZ: 2.2 (1.9, 2.5) pN, N = 1737; KIF1AP305L (1-393)-LZ: 0.7 (0.5, 0.8) pN, N = 637; WT KIF5B (1-420): 3.0 (2.4, 3.4) pN, N = 603; KIF5BP276 (1-420): 1.7 (1.1, 2.2) pN, N = 415. Statistical significance was determined using an unpaired Welch’s t test (****P < 0.0001). (F) Maximal force (“stall force”) sustained during a single run against load for a minimum time duration of 10 ms [3.1 (2.7, 3.5) pN, N = 405], 20 ms [3.1 (2.7, 3.4) pN, N = 381], 50 ms [3.2 (2.7, 3.4) pN, N = 262], and 100 ms [3.2 (2.8, 3.5) pN, N = 154], respectively. (G) Stall force of WT KIF5B (1-420) (≥20-ms criterion).

Supplementary Materials

  • Supplementary Materials

    A highly conserved 310 helix within the kinesin motor domain is critical for kinesin function and human health

    Aileen J. Lam, Lu Rao, Yuzu Anazawa, Kyoko Okada, Kyoko Chiba, Mariah Dacy, Shinsuke Niwa, Arne Gennerich, Dan W. Nowakowski, Richard J. McKenney

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