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Structure of the dynein-2 complex and its assembly with intraflagellar transport trains

Nature Structural & Molecular Biology volume 26, pages 823–829 (2019)Cite this article

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Abstract

Dynein-2 assembles with polymeric intraflagellar transport (IFT) trains to form a transport machinery that is crucial for cilia biogenesis and signaling. Here we recombinantly expressed the ~1.4-MDa human dynein-2 complex and solved its cryo-EM structure to near-atomic resolution. The two identical copies of the dynein-2 heavy chain are contorted into different conformations by a WDR60−WDR34 heterodimer and a block of two RB and six LC8 light chains. One heavy chain is steered into a zig-zag conformation, which matches the periodicity of the anterograde IFT-B train. Contacts between adjacent dyneins along the train indicate a cooperative mode of assembly. Removal of the WDR60−WDR34−light chain subcomplex renders dynein-2 monomeric and relieves autoinhibition of its motility. Our results converge on a model in which an unusual stoichiometry of non-motor subunits controls dynein-2 assembly, asymmetry, and activity, giving mechanistic insight into the interaction of dynein-2 with IFT trains and the origin of diverse functions in the dynein family.

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Fig. 1: Cryo-EM structure of the dynein-2 complex.
Fig. 2: DHC2 asymmetry and LIC3 binding.
Fig. 3: A block of intermediate and light chains controls dynein-2 asymmetry, oligomerization, and activity.
Fig. 4: The asymmetric structure of dynein-2 matches the periodicity of the IFT train.

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Data availability

Cryo-EM maps are available from the EMDB under accession codes EMD-4918 (dynein-2 tail domain) and EMD-4917 (dynein-2 motor domains). Coordinates are available from the RCSB Protein Data Bank under accession codes PDB 6RLB (dynein-2 tail domain), PDB 6RLA (dynein-2 motor domains), and PDB 6SC2 (dynein-2, docked into subtomogram average of the anterograde IFT-B train29 (EMDB-4303)). All other data supporting the conclusions of this manuscript are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank H. Mitchison, C. Moores, S. Webb, and G. Zanetti for comments on the manuscript; Diamond Light Source for cryo-EM facilities at the UK national electron bio-imaging centre (eBIC) supported by the Wellcome Trust, MRC and BBSRC; N. Lukoyanova, J. van Rooyen, A. Sielbert and D. Clare for help with cryo-EM data collection; and D. Houldershaw for computational support. This work was funded by Wellcome Trust and Royal Society (104196/Z/14/Z), BBSRC (BB/P008348/1), and Royal Society (RG170260) grants to A.J.R; Wellcome Trust (WT100387) and MRC grants (MC_UP_A025_1011) to A.P.C; and Wellcome Trust (079605/Z/06/Z) and BBSRC (BB/L014211/1) grants supporting cryo-EM equipment at Birkbeck.

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Authors and Affiliations

  1. Institute of Structural and Molecular Biology, Birkbeck, University of London, London, UK

    Katerina Toropova, Aakash G. Mukhopadhyay, Miroslav Mladenov & Anthony J. Roberts

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

    Ruta Zalyte & Andrew P. Carter

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  1. Katerina Toropova
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Contributions

K.T: investigation, methodology, visualization, writing of original draft. R.Z: investigation, methodology. M.M: investigation, methodology. A.G.M: investigation, writing - review and editing. A.P.C: investigation, methodology, funding acquisition, supervision, writing - review and editing. A.J.R: conceptualization, investigation, methodology, funding acquisition, supervision, visualization, writing of original draft.

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Correspondence to Anthony J. Roberts.

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Integrated supplementary information

Supplementary Figure 1 Recombinant expression and flexibility of the human dynein-2 complex.

(a) Assembly strategy for the dynein-2 expression plasmid. See Supplementary Table 1 for subunit nomenclature in different organisms. In mammals there are two paralogs for the light chains LC8 (1 and 2), RB (1 and 2), and TCTEX (1 and 3), in addition to the TCTEX-related protein TCTEX1D2. (b) Organisation of expression cassettes in the final plasmid. Each gene is flanked by a polH promoter and SV40 terminator. DHC2 contains an N-terminal ZZ tag and TEV cleavage site used for purification, followed by a SNAPf tag. (c) Size exclusion chromatogram of a dynein-2 complex preparation after affinity purification and TEV cleavage. V0; void volume. (d) SDS-PAGE of the peak fraction in (c). Mass spectrometry confirmed the presence of the indicated subunits. (e) Negative stain class averages reveal flexibility between the tail and motor domains of the dynein-2 complex. Particles are aligned on motor domains and classified to reveal varying tail domain positions. The angle between the C2 symmetry axis in the motor domains (dashed line) and the dimerization domain in the tail (blue dot) is shown. At high tail-motor angles, the TCTEX-TCTEX1D2 region of the tail is in close proximity to the motor domain. (f) Polar histogram plot of the angles between the tail and motor domains. A total of 1,120 particles, classified into 27 averages were analysed. Bins = 10 degrees. Arrows mark the median tail angle in isolated dynein-2 (gray) and the angle when dynein-2 is bound to the anterograde IFT-B train (maroon). (g) Overlay of the median tail angle in isolated dynein-2 and when IFT-bound. All subunits except DHC2 are omitted for clarity.

Supplementary Figure 2 Cryo-EM structure determination of dynein-2.

(a) Representative cryo-EM micrograph of dynein-2 particles (arrowheads; black – tail, white – motor domain). (b) Class averages of dynein-2 tail (top row) and motor domains (bottom row) in different orientations. (c) Fourier shell correlation (FSC) plots for the tail and motor domain reconstructions. Resolution at FSC=0.143 is marked. (d) Euler angle distribution of particles in the tail and (e) motor domain reconstructions. Cylinder height and color represent number of particles (blue to red = low to high). For the motor domain reconstruction, a hemisphere is shown as C2 symmetry was applied. (f) Tail and (g) motor domain reconstructions colored by local resolution as calculated by Relion. (h) – (j) Focused refinement of different regions in the tail domain. For each region, the mask applied to the reference during 3D refinement is shown on the left, with the refined map on the right.

Supplementary Figure 3 Map and model quality for the dynein-2 tail and motor domains.

(a) Cryo-EM density and models for the dynein-2 tail subunits. The overall tail map (Supplementary Fig. 2f) is shown, with the exception of the lower portion of DHC2-B and its associated LIC3, for which a map from focused refinement is shown (Supplementary Fig. 2j). See Fig. 1 for enlarged density examples. (b) Real-space correlation coefficient (CC) between the map and model for each subunit, calculated in Phenix (phenix.validation_cryoem). (c) Dynein-2 motor domain reconstruction (top) and refined atomic model in ribbon representation (bottom). AAA+ domains, linker and C-terminal domain (CTD) are labeled. (d) Example density in the motor domain map shown in mesh representation. Model is shown in stick representation. (e) Density at the AAA1–4 nucleotide binding sites suggests ADP at sites AAA1, 3 and 4 and ATP at AAA2.

Supplementary Figure 4 Dynein-2 intermediate chains, WDR60 and WDR34.

(a) Secondary structure elements and primary sequence of WDR34 (left) and WDR60 (right). Beta sheets are numbered according to the blade of the β-propeller domain to which they belong. LC8 binding β-sheets are shown as yellow arrows and RB-binding α-helices are in pink. *denotes regions of WDR34 involved in LC8 and RB binding as mapped by Tsurumi, Y. et al. (Mol. Biol. Cell. 30, 658-670, 2019). #denotes the region of WDR60 involved in TCTEX1D2 binding as mapped by Hamada, Y. et al. (Mol. Biol. Cell. 29, 1628–1639, 2018). Putatively disordered N-terminal region of WDR60 is not shown. (b) Cryo-EM density for the WDR34 and WDR60 ß-propellers with models in ribbon representation. Three large inserts in WDR60 (teal, arrowheads) allow unambiguous distinction from WDR34. (c) 3D classes showing varying position of density assigned to TCTEX1/TCTEX1D2 (orange).

Supplementary Figure 5 Contrasting architectures of dynein-2 and -1.

(a) Cryo-EM maps of dynein-2 tail and motor domains (solid) and an unsharpened map showing the flexible connection between them (transparent). (b) Cryo-EM maps of the dynein-1 tail [EMD-3703] and motor domains [EMD-3698] (solid) and entire molecule [EMD-3705] (transparent) (Zhang, K. et al. Cell. 169, 1303–1314, 2017). Dynein-2 has a strikingly asymmetric architecture compared to dynein-1, which allows dynein-2 to associate with the anterograde IFT-B train by matching its periodicity. DHC2TAIL is also shorter by one N-terminal bundle compared to the dynein-1 heavy chain, which uses this subdomain to engage the Arp filament of dynactin (Zhang, K. et al. Cell. 169, 1303–1314, 2017). (c, d) Comparison of the β-propeller domains of dynein-2’s heterodimeric intermediate chains, WDR60 and WDR34 (c) with dynein-1’s homodimeric intermediate chain (IC2) (d), following alignment based on RB. The WDR60 and WDR34 β-propellers are vertically offset and related by a ~90° rotation, which we propose drives the asymmetry of dynein-2, together with the block of three LC8 dimers which is also unique to dynein-2. (e, f) Comparison of dynein-2’s light-intermediate chain, LIC3 (e), with the dynein-1 light-intermediate chain, LIC2 (f) [EMDB-4171] (Urnavicius, L. et al. Nature. 554, 202–206, 2018). The dynein-1 light-intermediate chain has a long disordered C-terminal extension (dashed line) containing a helix that binds to dynein-1 cargo adaptors (Schroeder, C. M. et al. Elife. 3, e03351, 2014; Lee, I. G. et al. Nat Commun. 9, 986, 2018; Celestino, R. et al. PLoS Biol. 17, e3000100, 2019). In contrast, LIC3’s C-terminal region takes an opposite path and interacts with WDR60 within dynein-2.

Supplementary information

Supplementary Information

Supplementary Figures 1–5, Supplementary Tables 1 and 2, Supplementary Notes 1–3

Reporting Summary

Supplementary Video 1

Large-scale flexibility of the dynein-2 complex. Series of negative stain EM class averages of the dynein-2 complex showing the variation in tail position with respect to the aligned motor domains. Movie shows 27 class averages, ordered by tail angle, looped 6 times.

Supplementary Video 2

Cryo-EM structure of the dynein-2 complex reveals its marked asymmetry and unusual stoichiometry. Cryo-EM maps of the dynein-2 tail and motor domains are shown in surface representation, rotating about the y-axis, colored by subunit. Connecting density from an unsharpened map is shown as a transparent surface.

Supplementary Video 3

Dynein-2’s intermediate and light chains are required for motor auto-regulation. Microtubule gliding activity of the dynein- 2 holoenzyme and a construct lacking the intermediate and light chain sub-complex (∆IC-LC). Whereas the holoenzyme binds microtubules weakly and exhibits slow microtubule gliding, consistent with the majority of complexes being in an auto-inhibited state, ΔIC-LC drives rapid and continuous microtubule movement. See also Fig. 3e. Movies are shown at 45x real time.

Supplementary Video 4

Dynein-2’s asymmetric structure matches the periodicity of the anterograde IFT-B train. The cryo-EM structure of the dynein-2 complex docked into a ~40 Å sub-tomogram average of the IFT-B train from C. reinhardtii cilia (Jordan, M. et al. Nat. Cell Biol. 20, 1250–1255, 2018) (transparent surface representation). Dynein-2 complexes along the train are shown in alternating surface and cylinder representation for distinction. See also Fig. 4.

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Toropova, K., Zalyte, R., Mukhopadhyay, A.G. et al. Structure of the dynein-2 complex and its assembly with intraflagellar transport trains. Nat Struct Mol Biol 26, 823–829 (2019). https://doi.org/10.1038/s41594-019-0286-y

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