Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein and dynactin to distinct cargoes. Here we use electron microscopy and single-molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased towards recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two dyneins. We find that the shift towards a double dynein complex increases both the force and speed of the microtubule motor. Our 3.5 Å resolution cryo-electron microscopy reconstruction of a dynein tail–dynactin–BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side-by-side. Our work provides a structural basis for understanding how diverse adaptors recruit different numbers of dyneins and regulate the motile properties of the dynein–dynactin transport machine.
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We thank S. Scheres, X. Bai, K. Vinothkumar and R. Leiro for cryo-EM advice; S. Chen, G. McMullan, C. Savva, G. Cannone, J. Grimmett and T. Darling for technical support; S. Bullock for SNAPf–dynein (1-1074-GST); M. Yu for crystallography support and the European Synchrotron Radiation Facility (beamline ID29) for data collection; T. Croll for model building; S. Bullock, L. Passmore, S. Lacey and H. Foster for manuscript comments; and G. Lander for discussions. This work was funded by Wellcome Trust (WT100387) and MRC grants (MC_UP_A025_1011) to A.P.C.; and NIH (GM094522) and NSF (MCB-1055017, MCB-1617028) grants to A.Y.
The authors declare no competing financial interests.
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Extended data figures and tables
a, Cryo-EM reconstruction of the TDR complex analysed by ResMap52 and showing resolution distribution from 4 to 12 Å. b, The gold-standard Fourier shell correlation (FSC) curve of the 6.5 Å TDR map. c, Cryo-EM reconstruction of the TDH complex, showing resolution distribution from 4 to 12 Å. d, The gold-standard FSC curve of the 6.7 Å TDH map. e, Cryo-EM density for TDR low-pass filtered to 6.7 Å resolution (coloured according to cartoon) and to 20 Å (transparent outline). Density at the N terminus of BICDR1 is boxed. f, Cryo-EM density for TDH low-pass filtered to 6.7 Å (coloured according to cartoon) and to 20 Å (transparent outline) showing the putative Hook domain, an extension of the HOOK3 coiled coil ending in extra density near dynein-B (dashed box).
a, A one-cumulative frequency distribution plot showing run-lengths of DDB, DDR and DDH, with fit to a one-phase exponential decay. The decay constant (run length) and R2 value (least squares regression) of the fit are shown. We measured 785, 677 and 684 events for DDB, DDH and DDR, respectively, from microtubules of at least 20 μm in length from three chambers. b–f, Distribution of mean velocities of processive (unidirectional, minus-end-directed) events for DDB (n = 3,343) and DDR (n = 3,162) (b); DDB and DDH (n = 3,744) (c); active mutant dynein in complex with dynactin and BICD2 (mtDDB, n = 905) or BICDR1 (mtDDR, n = 1,183) (d); the colocalized mtDDR complexes containing both TMR–dynein tail and Alexa Fluor 647-full-length dynein (tail–dynein, n = 939) or Alexa Fluor 647-only complexes containing only full-length dynein (dynein-only, n = 1,004) (e); and all DDB complexes and complexes with both fluorophores, and hence two dyneins (colocalizers, n = 660) (f). Mean ± s.e.m. values were estimated by fitting the histograms to a Gaussian distribution (dashed lines).
a, Micrograph of the TDR complex (representative of 26,906 micrographs). b, Typical 2D-class averages of TDR in different orientations. c, The overall density map of TDR was analysed by ResMap, showing a resolution distribution from 3 to 8 Å. d, The gold-standard FSC curve of the overall TDR map. e, Mesh representation of 3.4 Å resolution density map of α helixes from dynein-B1 obtained by focused 3D classification and refinement. f, Sample density obtained by local sub-volume averaging, showing β strands from IC WD40.
Focused 3D classification and refinement procedures used in this study to improve density maps for dynein tails.
a, Secondary structure elements of dynein HC are matched against the primary sequence showing the NDD (purple) and the dynein helical bundles (blue; cyan; green; yellow; pale yellow; orange; red; pink). b, Secondary structure elements of IC. Extended N-terminal regions are coloured purple and other elements are coloured according to the blade of the WD40 domain to which they belong, except sheet β5, which associates with β30–32. c, Secondary structure elements of LIC, showing the globular domain helices and sheets (blue) and the two helices that pack against the HC (red). Jpred53 secondary structure predictions of features not seen in the electron microscopy map are shown in grey.
a, The dynein HC (yellow) interacts with the IC WD40 domain (blue) using bundles 4 and 5, with a helical segment (red cartoon) sitting in the WD40 central cavity. Dynein-A2 is shown. Interacting residues are shown as sticks (bottom panel), with HC residues in red and IC residues in green. b, Density map and model showing how the LIC (density and cartoon, blue) N- and C-terminal regions extend from the globular domain and pack against the HC (density, coloured by bundle number). Dynein-A2 is shown. c, ROBL1 (cartoon, light and dark pink) makes contacts with the IC N-terminal helices (cartoon, light and dark blue), which mediate the interaction between ROBL1 and the IC WD40 (surface). d, Representative density from the 1.9 Å resolution NDD crystal structure. e, Cartoon model of the NDD showing one chain in rainbow spectrum.
a, Conservation diagram showing sequence similarity between A2 and B1 interacting residues. Residues coloured white with red background are completely conserved, whereas residues coloured red show sequence similarity at that position. Residues at each interaction site are numbered below the alignment (A2 residues in yellow circles, B1 residues in red circles). These numbers label the accompanying cartoon to show the dynein chains that constitute each interaction. Alignment generated by ESPript54 (http://espript.ibcp.fr). b, Intermediate chain interactions showing connections between the IC of A1 and the HC of A2; the IC of A2 and the HC of B1; and the IC of B1 and the HC of B2. Interacting sites on each IC are shown as yellow spheres; sites on each HC are shown as red spheres. c, B1 (pink) contacts extra density (labelled, blue) adjacent to the BICDR1 coiled coil. The cartoon below shows the location of the area depicted (correspondingly coloured). d, Weak density connects the extra density with the LIC A2 helix 13 (blue). A cartoon representation of the area depicted is shown below.
a, The TDR structure (left) is compared to models of TDH (middle) and TDB (right). Although the paths of BICDR1 (yellow), HOOK3 (magenta) and BICD2 (orange) vary along the surface of dynactin (green surface), dynein-A HCs (light blue) bind at the same sites in each complex. b, Zoomed-in views of the barbed end of dynactin show that BICD2 adopts an upwards position to contact ARP1A (grey), whereas BICDR1 and HOOK3 adopt lower positions to bind dynein-B using the region coloured in red. The BICD2–ARP1A interaction site is highlighted in purple.
The full dynein tail/dynactin/BICDR1 complex (PDB:6F1T) is shown with the dynein tails (blue and red) and BICDR1 (yellow) shown in cartoon representation, and dynactin shown in surface representation (green). The complex is rotated, first to show the heavy chains in the grooves along dynactin’s filament, then to show interactions between the dynein tails and BICDR1. (MP4 12148 kb)
This video starts by focusing in on the two dynein tails from PDB 6F1T. Dynein interactions are then shown in sphere representation (colored according to Fig. 5b and Extended Data Fig. 7b). The complex is rocked to show the extensive interactions between dynein chains. (MP4 18435 kb)
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Urnavicius, L., Lau, C., Elshenawy, M. et al. Cryo-EM shows how dynactin recruits two dyneins for faster movement. Nature 554, 202–206 (2018). https://doi.org/10.1038/nature25462
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