Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements

Abstract

Cilia are multifunctional organelles that are constructed using intraflagellar transport (IFT) of cargo to and from their tip. It is widely held that the retrograde IFT motor, dynein-2, must be controlled in order to reach the ciliary tip and then unleashed to power the return journey. However, the mechanism is unknown. Here, we systematically define the mechanochemistry of human dynein-2 motors as monomers, dimers, and multimotor assemblies with kinesin-II. Combining these data with insights from single-particle EM, we discover that dynein-2 dimers are intrinsically autoinhibited. Inhibition is mediated by trapping dynein-2's mechanical 'linker' and 'stalk' domains within a novel motor–motor interface. We find that linker-mediated inhibition enables efficient transport of dynein-2 by kinesin-II in vitro. These results suggest a conserved mechanism for autoregulation among dimeric dyneins, which is exploited as a switch for dynein-2's recycling activity during IFT.

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Figure 1: Intraflagellar transport motors and constructs used in this study.
Figure 2: Monomeric dynein-2 motor domains power fast microtubule gliding.
Figure 3: Dimerization inhibits dynein-2 motor domains.
Figure 4: Dynein-2's linker and stalk are trapped within a novel motor–motor interface.
Figure 5: Untrapping dynein-2 dimers rescues their motility.
Figure 6: Assembly and motility of dynein-2 and kinesin Kif3 in multimotor arrays.
Figure 7: Model for dynein-2 regulation during IFT.

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Acknowledgements

We thank C. Moores, G. Zanetti and A. Osborne for critical comments on the manuscript; M. Williams, S. Reck-Peterson and members of the Birkbeck EM group for advice; S. Nofal, S. Miah, and L. Stejskal for initial experiments; and A. Carter (MRC-LMB, Cambridge) for plasmids. This work was supported by a Sir Henry Dale Fellowship to A.J.R. from the Wellcome Trust and Royal Society [104196/Z/14/Z].

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Contributions

K.T. and A.J.R. conducted biochemical and TIRF experiments. K.T. performed electron microscopy experiments. M.M., K.T., and A.J.R. generated and purified constructs. K.T. and A.J.R. analyzed data and wrote the paper.

Corresponding author

Correspondence to Anthony J Roberts.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Influence of microtubule length and buffer conditions on dynein-2 microtubule gliding.

(a) Kymographs of microtubule gliding at 4 nM Dyn2motor. Microtubule lengths are shown below. (b) Plot of gliding velocity as a function of microtubule length at 4 nM input concentration of Dyn2motor. N = 63 microtubules. Microtubule length and velocity are correlated with a Spearman coefficient of 0.56 (p < 0.0001). (c) Plot of mean microtubule gliding velocity (± s.d.) at different dynein-2 motor input concentrations in buffer lacking 50 mM KCl. Slower microtubule gliding velocities were seen for all three dynein-2 constructs in the absence of KCl (compare with Fig. 2e, 3d and 5e). Number of microtubules analyzed per concentration as follows: Dyn2motor 0.2 nM (32), 0.5 nM (46), 2 nM (47), 5 nM (43), 20 nM (48), 200 nM (90), GST-Dyn2motor 0.2 nM (70), 2 nM (32), 10 nM (42), 20 nM (51), 100 nM (46), 200 nM (51), GST-Dyn2(DQR)motor 0.2 nM (35), 1 nM (56), 2 nM (52), 10 nM (51), 20 nM (52), 200 nM (58). Fitted values (± standard error of the fit): Dyn2motor Vmax = 219.3 ± 3.3 nm/s, f = 0.2 ± 0.01, GST-Dyn2motor Vmax = 168.0 ± 2.1 nm/s, f = 0.1 ± 0.004, GST-Dyn2(DQR)motor Vmax = 259.0 ± 2.7 nm/s, f = 0.3 ± 0.01.

Supplementary Figure 2 Impact of nucleotide, salt, and mutagenesis on dynein-2 dimer architecture.

(a) EM micrographs GST-Dyn2motor dimers in different nucleotide and buffer conditions. High salt, 500 mM KCl. GST-Dyn2motor domains appear stacked in ADP.Vi and separated in other conditions. GST-Dyn2motor domains are also stacked in ATP conditions (micrograph shown in Fig. 3c). Arrow, particularly well-resolved GST-Dyn2motor dimer in the separated configuration in which stalks are visible in the raw micrograph. (b) EM micrograph GST-Dyn2(DQR)motor dimers in ATP. (c) Histograms showing the distribution of 2D motor–motor distances in EM class averages of GST-Dyn2motor and GST-Dyn2(DQR)motor. Total number of molecules analyzed: GST-Dyn2motor ATP (917), ADP.Vi (1160), no-nucleotide (890), ADP (853), ATP High salt (846), GST-Dyn2(DQR)motor ATP (891). The GST-Dyn2motor distributions in ATP and ADP.Vi conditions show a peak at low motor–motor separation indicative of stacking. Stacking is largely abolished in GST-Dyn2(DQR)motor.

Supplementary Figure 3 Linker-mediated stacking model and consistency with dynein-1 and dynein-2 data.

(a) Upper panel: Model of the autoinhibited state of dynein-2, derived from PDB 4RH7 (Schmidt, H. et al., Nature. 518, 435-8, 2015). The linker domains (magenta) are trapped in the motor–motor interface, while the C-terminal domains (CTDs) are on the periphery of the dimer and do not interact. All motor–motor interfaces are labeled. Lower panel: previous model of the cytoplasmic cytoplasmic dynein-1 phi particle (Torisawa, T. et al., Nature Cell Biology. 16, 1118-24, 2014), derived from PDB 3VKG (Kon, T. et al., Nature. 484, 345-50, 2012). The motor domains interact via their CTDs and the linker domains are free to move on the periphery of the dimer. (b) Enlarged views of dynein-2 interfaces in the new model. Amino acids involved in inter-motor domain interactions are labeled. (c) Top row, EM averages of GST-Dyn2motor in ATP and ADP.Vi conditions. Bottom row, highest scoring projections of the new model based on cross correlation. GST-Dyn2motor shows preferred orientations on the EM support, which are matched by the new model with Euler angles shown (ϕ, θ; SPIDER convention). GST/SNAPf density is labeled with an arrowhead in the EM average and is absent in projections. (d) Equivalent analysis carried out for human cytoplasmic dynein-1 holoenzyme in the phi particle configuration. The tail and associated subunits of the holoenzyme are labeled with an arrow in the EM average. Scale bar, 10 nm. The linker-mediated stacking model is consistent with class averages of both dynein-1 and dynein-2.

Supplementary Figure 4 DQR mutations have little or no impact on dynein-2 monomer activity.

(a) Sequence diagrams of dimeric GST-Dyn2(DQR)motor, and the Dyn2(DQR)motor construct used to assess if the DQR mutations impact activity in the context of a dynein-2 monomer. (b) Size-exclusion chromatograms. Dyn2(DQR)motor was normalized to the peak value of GST-Dyn2(DQR)motor. (c) Velocity of microtubule gliding at 1 and 20 nM concentrations of Dyn2motor and Dyn2(DQR)motor. Black and pink lines show mean ± s.d. Number of microtubules analyzed per concentration: Dyn2motor 1 nM (39), 20 nM (56), Dyn2(DQR)motor 1 nM (29), 20 nM (49). (d) Microtubule-stimulated ATPase activity of Dyn2(DQR)motor (Dyn2motor values from Fig. 2f shown in gray for comparison). Experiments were carried out in triplicate, mean values ± s.d. are shown. Fitted values (± standard error of the fit): kcat = 4.7 ± 0.3 s-1, kbasal = 1.6 ± 0.1 s-1, Km(MT) = 8.0 ± 2.2 μM.

Supplementary Figure 5 Purification and motility of Kif3 Δtail.

(a) Sequence diagrams of Kif3 and Kif3 Δtail, in which mutations that prevent autoinhibition are introduced and putatively disordered C-terminal regions are removed. Yellow box, SNAPf tag. (b) Size-exclusion chromatogram of Kif3 Δtail and schematic of the construct. (c) SDS-PAGE of Kif3 ∆tail after the final purification step. (d) Plot of mean microtubule gliding velocity (± s.d.) at different Kif3 Δtail input concentrations. Number of microtubules analyzed per concentration: 0.5 nM (50), 0.7 nM (48), 2 nM (43), 5 nM (48), 20 nM (50), 60 nM (47). Fitted values (± standard error of the fit): Vmax = 537.0 ± 2.0 nm/s, f = 0.99 ± 0.002. (e) Kymograph showing single-molecule motility of Kif3 Δtail labeled with Alexa647 via its SNAPf tag. (+) and (–) indicate microtubule polarity. (f) Velocity histogram of Kif3 Δtail single molecules (N = 311 molecules).

Supplementary Figure 6 Assembly and motility of synthetic trains bound with dynein-2.

(a) Gel shift assay showing migration of DNA origami chassis samples with three (3x) or seven (7x) attachment sites and no dynein (‘none’), GST-Dyn2motor (‘WT’), or GST-Dyn2(DQR)motor (‘DQR’) bound. (b,c) Kymographs showing behavior of DNA origami assemblies with (b) seven GST-Dyn2motor and (c) seven GST-Dyn2(DQR)motor sites. (+) and (–) indicate microtubule polarity.

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Supplementary Figures 1–6 (PDF 1380 kb)

Supplementary Data : Model of dynein-2 in its autoinhibited state.

Coordinates for the linker-stacking model of dynein-2 autoinhibition, derived from PDB 4RH7. (TXT 3609 kb)

Impact of motor concentration on dynein-2 microtubule gliding.

Microtubule gliding powered by Dyn2motor at different concentrations. See also Fig. 2d. Videos are shown at 24X real time. (MOV 4884 kb)

Dynein-2 dimer architecture in different nucleotide conditions.

Class averages of GST-Dyn2motor in no nucleotide and 1 mM ATP conditions. See also Fig. 4b. Videos show 38 class averages in each condition, looped 4 times. (MOV 3134 kb)

Pairs of dynein-2 motor domains in crystallo match the architecture of isolated dimers.

Analysis of PDB 4RH7 crystal lattice reveals pairs of monomeric dynein-2 motor domains matching the stacked architecture of Dyn2motor dimers observed by single-particle electron microscopy. (MOV 8736 kb)

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Toropova, K., Mladenov, M. & Roberts, A. Intraflagellar transport dynein is autoinhibited by trapping of its mechanical and track-binding elements. Nat Struct Mol Biol 24, 461–468 (2017). https://doi.org/10.1038/nsmb.3391

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