Structure of dynein-dynactin on microtubules shows tandem adaptor binding

Abstract

Cytoplasmic dynein is a microtubule motor that is activated by its cofactor dynactin and a coiled-coil cargo adaptor^1-3. Up to two dynein dimers can be recruited per dynactin, and interactions between them affect their combined motile behaviour^4-6. Different coiled-coil adaptors are linked to different cargos^7,8, and some share motifs known to contact sites on dynein and dynactin^4,9-13. 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.

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). 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 p150^Glued 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 linker. c, A close-up view of the linker-AAA2 interaction overlaid with the crystal structures of D. discoideum dynein-ADP (PDB: 3VKG) and S. cerevisiae dynein-AMPPNP (PDB: 4W8F) aligned at the linker. The inset shows the crystal structure of S. cerevisiae dynein-Apo (PDB: 4AKG), 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), and S. cerevisiae dynein-AMPPNP (right; PDB: 4W8F) 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 ofD. discoideum dynein-ADP (left; PDB: 3VKG) and S. cerevisiae dynein-AMPPNP (right; PDB: 4W8F) 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. 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). 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. 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), withlower 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), aligned at dynactin. The pointed end interaction sites are labelled 1 to 4. 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, TRAK, and Hook3 are shown based on our analyses and previous predictions. 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.

Figure 1. Cryo-EM structure of dynein-dynactin-BICDR on MTs.

a, Example of a MT before and after subtraction from cryo-EM micrographs (n = 645,193 MTs on micrographs from 15 datasets). Representative 2D averages of particles from each case are shown on the right (26,158 and 21,696 particles, respectively). b, Gallery of locally processed density maps, starting with the consensus structure in the middle. c, Composite density map of dynein-dynactin-BICDR overlaid on the 13 protofilament MT. d, Density map of the dynein motor domain overlaid on the full motor (left) with the subdomains highlighted (right). e, Density in the nucleotide pocket of AAA3 with AMPPNP docked. f, Density in the nucleotide pocket of AAA1 with ADP docked.

Figure 2. Interactions of the motor domains.

a, Top view of the density map of the aligned state (left) and composite map overlaid onto the MT (right). Arrows represent the direction of travel. b, Top view of the density map of the staggered state (left) and composite map overlaid on the MT (right). c, A molecular surface representation of adjacent motor domains in the staggered state, only showing the linker of the righthand motor. Overlaid are ribbon representations of the C-terminal domain (CTD), AAA3, and linker. The hinge in the linker is also highlighted (triangle and dotted line), and arrows point at the relative position of the ring and tail. d, Top view of a molecular surface representation of dynein in the staggered state. e, Molecular surface representation of the motor domain of dynein-A2 (pink) and the LIC of dynein-B1 (green).

Figure 3. Two BICDRs scaffold the dynein-dynactin complex.

a, Top view of the density map of the dynactin and dynein-tail region (the density for dynactin is hidden). The N-terminal motifs (CC1 box and HBS1) are highlighted on each BICDR. b, Side view of the pointed end density map showing both BICDRs. c, A close up of the N-terminus of BICDR-A, showing the density around the CC1 box. A gaussian filter was applied to better show the LICs. d, A close up of the interaction between BICDR-A and dynein-A2, highlighting the HBS1 conserved residues (Q150/H153) and C-terminal E165/E172.

Figure 4. Pointed end interactions of BICDR-A/B.

a, The density map of the pointed end showing BICDR-A and BICDR-B contacting four distinct sites. The break in the coiled-coil density is highlighted (triangle). b, The density map of the pointed end showing the ordered alpha helical density at site 4. Also highlighted is the loop from p25 representing site 3. c, A molecular surface representation of the pointed end p25 subunit coloured by hydrophobicity (orange is hydrophobic, teal is hydrophilic). Overlaid is a ribbon representation of BICDR-A’s Spindly motif, highlighting the conserved residues L347 and E350.