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Structure of dynein–dynactin on microtubules shows tandem adaptor binding

Nature volume 610, pages 212–216 (2022)Cite this article

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A Publisher Correction to this article was published on 24 October 2022

This article has been updated

Abstract

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor1,2,3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour4,5,6. Different coiled-coil adaptors are linked to different cargos7,8, and some share motifs known to contact sites on dynein and dynactin4,9,10,11,12,13. There is limited structural information on how the resulting complex interacts with microtubules and how adaptors are recruited. Here we develop a cryo-electron microscopy processing pipeline to solve the high-resolution structure of dynein–dynactin and the adaptor BICDR1 bound to microtubules. This reveals the asymmetric interactions between neighbouring dynein motor domains and how they relate to motile behaviour. We found that two adaptors occupy the complex. Both adaptors make similar interactions with the dyneins but diverge in their contacts with each other and dynactin. Our structure has implications for the stability and stoichiometry of motor recruitment by cargos.

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Fig. 1: Cryo-EM structure of dynein–dynactin–BICDR on MTs.
Fig. 2: Interactions of the motor domains.
Fig. 3: Two BICDRs scaffold the dynein–dynactin complex.
Fig. 4: Pointed-end interactions of BICDR-A and BICDR-B.

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Data availability

Atomic coordinates and cryo-EM maps have been deposited in the Protein Data Bank or Electron Microscopy Data Bank (EMDB), respectively, under accession codes 7Z8F and 14549 (composite dynein–dynactin–BICDR structure), 7Z8G and 14550 (dynein motor domain), 7Z8H and 14551 (dynein AAA1–3), 7Z8I and 14552 (barbed end–BICDR-A), 7Z8J and 14553 (BICDR-A–dynein-A2), 7Z8K and 14555 (BICDR-B–dynein-A1), 7Z8L and 14556 (dynein motor domain–LIC), 7Z8M and 14559 (pointed end–BICDR-A), and 15396 (consensus map). Other atomic coordinates used in this study for alignments and model building are available in the Protein Data Bank (2PG1, 3VKG, 4AKG, 4W8F, 5NUG, 6F1T, 6RZB, 6PSE, 6ZNL and 7K58). The cryo-EM map of dynein-tail–dynactin–BICDR1 used for comparisons is available in the Electron Microscopy Data Bank (4168). The protein sequences used for sequence alignment and AlphaFold predictions are available at the Universal Protein Resource (UniProt) under accession codes Q13409, O43237, Q9UJW0, O00399, Q9BTE1, I3LHK5, A0JNT9, Q8TD16, Q96EA4, Q9UPV9, Q86VS8, O75154, Q9BSW2, Q9UPT6, Q96NA2, Q14204, Q6ZP65, A0A140LGI1, Q8BUK6 and Q6GQ73.

Code availability

Custom scripts, including the Starparser package, are available at https://github.com/sami-chaaban (https://doi.org/10.5281/zenodo.6792794, https://doi.org/10.5281/zenodo.6792805 and https://doi.org/10.5281/zenodo.6792801).

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Acknowledgements

We thank S. Scheres for help with micrograph-level signal subtraction in Relion; C. K. Lau for helpful discussions; the MRC Laboratory of Molecular Biology Electron Microscopy Facility for access and support of electron microscopy sample preparation and data collection; J. Grimmett and T. Darling for providing scientific computing resources; H. E. Foster and C. Ventura Santos for help with cryo-ET; and F. Abid Ali, K. Singh and C. K. Lau for critical reading of the manuscript. This work was supported by Wellcome (210711/Z/18/Z), the Medical Research Council, as part of UK Research and Innovation (MRC file reference number MC_UP_A025_1011), and the EMBO Postdoctoral Fellowship (ALTF 334-2020) to S.C. For the purpose of open access, the author has applied a CC BY public copyright license to any author-accepted manuscript version arising.

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  1. Division of Structural Studies, Medical Research Council Laboratory of Molecular Biology, Cambridge, UK

    Sami Chaaban & Andrew P. Carter

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  1. Sami Chaaban
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Contributions

S.C. performed the experiments and analysis and prepared the figures. S.C. and A.P.C. conceived the project and wrote the manuscript.

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Correspondence to Andrew P. Carter.

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Extended data figures and tables

Extended Data Fig. 1 MT subtraction from cryo-EM micrographs of dynein-dynactin-BICDR on MTs.

a, An example micrograph out of n = 66,800 that had microtubules suitable for subtraction. A pseudo-flat-field correction has been applied to normalize the intensity across the micrograph for visualization purposes (i.e. dividing the image by a gaussian-blurred copy). b, Overview of the processing pipeline to subtract MTs from cryo-EM images in order to thoroughly pick and accurately align dynein-dynactin-BICDR complexes. c, The density maps of the 12, 13, and 14 protofilament (pf) MTs. Not shown are the 11, 15, and 16 pf MTs. d, An annotated micrograph showing picked particles that were kept (black) or rejected (red) based on their proximity to the MTs.

Extended Data Fig. 2 Processing pipeline for single-particle analysis of the dynein-dynactin-BICDR complex.

(T = Tau fudge, C = number of classes). 3D classifications are without alignment unless otherwise specified. All defocus, magnification, and beam-tilt refinements were immediately followed by a 3D refinement (not shown). Plots show the gold standard Fourier shell correlation. The dotted horizontal line shows the 0.143 cut-off. An angular distribution plot is shown for the consensus refinement of the whole dataset on a Mollweide projection.

Extended Data Fig. 3 Overview of the dynein-dynactin-BICDR complex.

a, The composite density map of dynein-dynactin-BICDR overlaid on the reconstructed 13 protofilament MT, showing the position of individual dynein motor domains (dynein-A1/2, dynein-B1/2), with their tails extending towards dynactin. b, A pseudo-molecular surface representation of a dynein dimer, showing the LICs, intermediate chains (ICs) and light chains (LCs). The model was generated from our structure and the published IC/LC8/Tctex crystal structure (PDB: 2PG1)74. Additional flexible regions were added manually. c, The composite density map of the complex shown from behind, where dynein’s tails can be seen sitting in the grooves of dynactin’s Arp1 filament. d, A molecular surface representation of our model of dynactin viewed from the back, showing the position of dynactin’s Arp1 filament and barbed/pointed ends relative to the dynein tails and BICDRs. e, A 3D classification result showing density connecting the shoulder domain to a globular density near dynein-A1, which may represent the Inter-Coiled Domain (ICD) of p150Glued with adjacent coiled-coils (CC1 and CC2).

Extended Data Fig. 4 Conformation of the dynein motor domain.

a, Side view of the four motor domains from both major configurations (aligned and staggered) displaying a straight linker (dotted black line). The extra density on dynein-A2 in the staggered state (dotted green line) represents the LIC of dynein-B1. b, Front view of a ribbon representation of the motor domain showing only the linker (purple), AAA1 (blue), AAA4 (yellow), AAA5 (orange) and C-terminal domain (grey). The inset shows the conserved F3629 in AAA5 binding the linker75. c, A close-up view of the linker-AAA2 interaction overlaid with the crystal structures of D. discoideum dynein-ADP (PDB: 3VKG)29 and S. cerevisiae dynein-AMPPNP (PDB: 4W8F)15 aligned at the linker. The inset shows the crystal structure of S. cerevisiae dynein-Apo (PDB: 4AKG)75, which lacks nucleotides in AAA1 and AAA3 and the linker is undocked from AAA2. d, The domain movements in the nucleotide pocket of AAA3 represented by arrows after alignment of our structure (Motor-MT) to the crystal structure of D. discoideum dynein-ADP (left; PDB: 3VKG)29, and S. cerevisiae dynein-AMPPNP (right; PDB: 4W8F)15 at AAA3L. e, The domain movements in the nucleotide pocket of AAA1 represented by arrows after alignment of our structure (Motor-MT) to the crystal structure of D. discoideum dynein-ADP (left; PDB: 3VKG)29 and S. cerevisiae dynein-AMPPNP (right; PDB: 4W8F)15 at AAA1L.

Extended Data Fig. 5 Motor interactions and heterogeneity.

a, Number of particles from the staggered and aligned states on different MT protofilament numbers (79,435 and 83,450 total particles, respectively). The plot shows the mean of n = 15 datasets representing independently prepared cryo-EM grids. Error bars show the 95% confidence interval (Mann Whitney U Test, two-sided; P = 0.43, 0.31, 0.24, 0.14, and 0.38, respectively) (ns = not significant). b, Front view of a 3D classification result of dynein-A in the staggered state showing a small subset of particles with parallel stalks (right), compared to the majority of particles with crossed stalks (left). A ribbon representation of the motor domain is placed in the density map to show the orientation of the stalks. c, A 3D classification result that includes the MT wall, showing the orientation of the protofilaments as well as density for the stalks of dynein-A. d, A closeup view of the interaction between dynein-A2 and the LIC of dynein-B1, highlighting the three potential interaction sites on the LIC. e, The interaction between dynein motor domains in the aligned (left) and staggered (right) states is shown as a molecular surface representation and density map, where the linker is coloured darker. The triangles and dotted lines highlight the hinge in the linker. f, Tomograms of individual dynein-dynactin-BICDR complexes on MTs in the aligned and staggered states. The dynein and dynactin densities have been coloured pink/purple and blue, respectively. g, An example tomogram where dynein-A1 is shifted away from dynein-A2. h, An example tomogram where there is a large separation between dynein-B1 and B2.

Extended Data Fig. 6 Adaptor arrangements and interactions.

a, A mass photometry result of purified BICDR on its own, showing a major peak at ~127 kDa (expected molecular weight of the dimer is 130 kDa) and a minor peak at 260 kDa (n = 1). b, The density maps of BICDR-A and BICDR-B showing the bulky density at W166 and the location of the HBS1 motif residues QEKH near dynein-A2 and dynein-A1, respectively. c, The interaction interface of the two BICDRs is shown based on their registry in the structure. Lowercase letters refer to the position in the heptad repeat of the coiled-coil, predicted with LOGICOIL76. The red asterisk at K234 shows the offset between the two BICDRs. d, A 3D classification result from the dynein-tail/dynactin/BICDR dataset which shows density for BICDR-B. e, The density map of dynein-tail/dynactin/BICDR (left) (EMD: 4168)4 and our consensus density map from the high-magnification dataset (right). Both maps were low-pass filtered to 10 Å and are shown at contour levels where the dynactin density is similar. f, A 3D classification of dynein-tail/dynactin/Hook34 showing that the second coiled-coil belongs to a second Hook3.

Extended Data Fig. 7 CC1 box interactions and the HBS1 motif of Hook3.

a, The LIC helix after fitting to the density on the inside face of the BICDR-A CC1 box relative to the registry of BICDR in our structure (left), highlighting the conserved A116, A117, and G120 of the motif. On the right is a similar view of the BICD2-LIC crystal structure (PDB: 6PSE)9. b, A 3D classification result showing the LIC of dynein-A2 connecting to the inside face of BICDR-A. c, A 3D classification result showing the LIC of dynein-A2 connecting to the inside face of BICDR-B. d, Sequence alignment of the HBS1 motif of BICDR (annotated as BICL1) and Hook3, highlighting the conserved residues and C-terminal glutamates (black circles). The UniProt codes are indicated on the left. e, An Alphafold prediction of two copies of Hook3 (fragment 172-287), the dynein heavy chain (fragment 576-864), and intermediate chain (fragment 226-583). In the middle, the PAE is displayed on the models relative to H200 (yellow), with lower values representing higher confidence. The full PAE plot is shown on the right, with the arrow pointing at H200.

Extended Data Fig. 8 Pointed end interactions and adaptor families.

a, An overlay of our structure with the previous dynein-tail/dynactin/BICDR structure (PDB: 6F1T)4, aligned at dynactin. The pointed end interaction sites are labelled 1 to 413. b, An Alphafold prediction of the pointed end complex (Arp11, p25, p27, and p62) and a C-terminal fragment of BICDR (205-394) that includes the Spindly motif. The models are coloured based on the predicted aligned error (PAE) at L347 of BICDR (yellow), with lower values representing higher confidence. The full PAE plot is also shown with the arrow pointing at L347. c, Alphafold predictions of cargo adaptors that have been manually linearized such that the coiled-coils are parallel to each other (i.e. each individual model was manually rotated at the disordered loops). White triangles depict breaks in the coiled-coil prediction preceding the Spindly motifs. The predictions are of full-length proteins unless otherwise stated. LIC-binding motifs (CC1 box, Hook domain, EF hand, RH1 domain), HBS1s, and Spindly motifs are coloured according to the legend. Only the HBS1s of BICDR, BICD2, Spindly, TRAK1, and Hook3 are shown based on our analyses and previous predictions10. The orientations are C-terminus to N-terminus to match the orientation in other figures. d, The predicted local distance difference tests (pLDDTs) (left) from one chain of each of the cargo adaptors around the Spindly motif (highlighted in red) and the full PAE plots (right). White triangles point to the locations of the predicted breaks in the coiled-coils.

Extended Data Table 1 Cryo-EM data collection, refinement, and validation statistics of the dynactin/dynein-tail regions
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Extended Data Table 2 Cryo-EM data collection, refinement, and validation statistics for the dynein motor domain regions
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Chaaban, S., Carter, A.P. Structure of dynein–dynactin on microtubules shows tandem adaptor binding. Nature 610, 212–216 (2022). https://doi.org/10.1038/s41586-022-05186-y

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