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Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport

Nature Structural & Molecular Biology volume 30, pages 1365–1379 (2023)Cite this article

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Abstract

The lissencephaly-related protein LIS1 is a critical regulator of cytoplasmic dynein that governs motor function and intracellular localization (for example, to microtubule plus-ends). Although LIS1 binding is required for dynein activity, its unbinding prior to initiation of cargo transport is equally important, since preventing dissociation leads to dynein dysfunction. To understand whether and how dynein–LIS1 binding is modulated, we engineered dynein mutants locked in a microtubule-bound (MT-B) or microtubule-unbound (MT-U) state. Whereas the MT-B mutant exhibits low LIS1 affinity, the MT-U mutant binds LIS1 with high affinity, and as a consequence remains almost irreversibly associated with microtubule plus-ends. We find that a monomeric motor domain is sufficient to exhibit these opposing LIS1 affinities, and that this is evolutionarily conserved between yeast and humans. Three cryo-EM structures of human dynein with and without LIS1 reveal microtubule-binding induced conformational changes responsible for this regulation. Our work reveals key biochemical and structural insight into LIS1-mediated dynein activation.

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Fig. 1: The microtubule-unbound dynein mutant is tightly bound to Pac1, Bik1, and microtubule plus-ends in cells.
Fig. 2: The dynein motor domain is sufficient for microtubule-binding-induced allostery.
Fig. 3: Mass photometric analysis of Pac1–dyneinMOTOR binding.
Fig. 4: Mass photometric analysis of human LIS1–dyneinMOTOR binding.
Fig. 5: Cryo-EM structures of human dyneinMOTORMT-B and a LIS1-bound dyneinMOTORMT-U.
Fig. 6: Cryo-EM structure of human dyneinMOTORMT-U alone.
Fig. 7: Changes at sitering account for reduced LIS1 and Pac1 binding affinity.

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Data availability

Models and cryo-EM maps have been deposited in the PDB and EMD as follows: PDB 8FCY and EMD-28999 (dyneinMOTORMT-B in ATP), PDB 8FD6 and EMD-29003 (dyneinMOTORMT-U in ATP and Vi), PDB 8FDT and EMD-29012 (dyneinMOTORMT-U + LIS1 in ATP and Vi), and PDB 8FDU and EMD-29014 (dyneinMOTORMT-U + LIS1 in ATP and Vi, locally refined at AAA3-AAA5 with 2 LIS1s). Residues labeled in the figures for human dynein are based on the Cytoplasmic Dynein-1 Heavy Chain 1 sequence (Q14204). All of the plasmids, yeast strains, datasets and raw video files that were generated during and/or analyzed during this study are available from the corresponding authors upon request. All data used to generate plots throughout the manuscript are included as Source Data files, as are raw images of uncropped gels. PDB models used throughout this study include 5NUG (ref. 3), 4RH7 (ref. 37), 7MGM (ref. 39), 8DYU (ref. 41), 7ZG8 (ref. 38), 3VKG (ref. 79), 4AKG (ref. 80), 4W8F (ref. 81), and 1UUJ (ref. 82). Source data are provided with this paper.

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Acknowledgements

We are very grateful to Janelia Research Campus for providing fluorescent Halo and SNAP dyes, A. Carter and S. Chabaan for valuable discussions (and for sharing data that was unpublished at the time), and to members of the Markus and DeLuca laboratories for valuable discussions. This work was funded by the NIH/NIGMS (R35GM139483 to S.M.M., and R35GM142959 to K.Z.) and in part by a Collaboration Development Award Program from the Pittsburgh Center for HIV Protein Interactions (U54AI170791 to K.Z.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of this manuscript. We would like to thank K. Zhou and J. Lin for the help with cryo-EM data collection at Yale ScienceHill-Cryo-EM facility. The Yale Cryo-EM Resource is funded in part by the NIH grant S10OD023603. We thank L. Wang, J. Kaminsky, and G. Hu at the Laboratory for BioMolecular Structure (LBMS) for help with cryo-EM data collection. The LBMS is supported by the DOE Office of Biological and Environmental Research (KP1607011).

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Author notes

  1. These authors contributed equally: William D. Ton, Yue Wang, Pengxin Chai.

  2. These authors jointly supervised this work: Kai Zhang, Steven M. Markus.

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA

    William D. Ton, Cisloynny Beauchamp-Perez, Nicholas T. Flint, Lindsay G. Lammers & Steven M. Markus

  2. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Yue Wang, Pengxin Chai, Hao Xiong & Kai Zhang

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Contributions

S.M.M. designed the study. W.D.T., C.B.-P., N.T.F., L.G.L., and S.M.M. purified yeast proteins, and W.D.T., C.B.-P., and S.M.M. purified human proteins. L.G.L. performed ATPase assays with MT-U and MT-B proteins. W.D.T., N.T.F, and S.M.M. performed in vitro binding assays. W.D.T., N.T.F., L.G.L., and S.M.M. performed and analyzed live-cell microscopy. Y.W. and P.C. acquired and analyzed cryo-EM data, and built PDB models with support from K.Z. H.X., and Y.C. performed molecular dynamics simulations. S.M.M., Y.W., P.C., L.G.L., and S.M.M. generated figures. S.M.M. wrote the manuscript. W.D.T, Y.C., P.C., K.Z., and S.M.M. edited and revised the manuscript. S.M.M. and K.Z. acquired funding.

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Correspondence to Kai Zhang or Steven M. Markus.

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Nature Structural & Molecular Biology thanks Arne Gennerich, Clinton Lau and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Design strategy and validation of microtubule-bound and -unbound dynein mutants.

(a) Cartoon and structural depiction of conformational change that takes place within the coiled-coil (CC) stalk and microtubule-binding domain (MTBD) upon microtubule binding. Comparison of a high resolution microtubule-bound dynein MTBD (6RZB)30 and a crystal structure of a microtubule unbound MTBD (3ERR)32 reveals an upward shift of helix 1 (H1) as a result of microtubule binding. This results in a consequent upward shift of CC1 with respect to CC2. (b) Design strategy to generate constitutive microtubule-unbound and -bound dynein mutants. The stable coiled-coil from seryl tRNA synthetase (SRSCC) was used to replace the entire dynein MTBD plus short regions of CC1 and CC2. The MT-B mutant has 4 additional residues in CC1 with respect to the MT-U mutant (corresponding to one helix turn), resulting in a presumed upward shift of CC1 compared to CC2. (c) Plot (mean ± SD, along with individual data points) depicting ATPase levels for artificially dimerized dynein motor domain fragments (GST-dyneinMOTOR; n = 2 independent replicates for each). Note the MT-U mutant closely reflects the wild-type dynein motor in the absence of microtubules, while the MT-B mutant matches that of wild-type plus a saturating concentration of microtubules (2 µM)58. (d) Representative images (fluorescence for dyneinMOTOR fragments, and interference reflection microscopy for microtubules) showing the ability of wild-type, but not dyneinMT-U or dyneinMT-B, to bind microtubules (images are representative of 2 independent replicates). Coverslip-immobilized microtubules were incubated with 50 nM of indicated dyneinMOTOR fragments (labeled via C-terminal HaloTagJFX646) prior to imaging.

Source data

Extended Data Fig. 2 Additional mass photometric analysis of Pac1- and LIS1-dyneinMOTOR binding.

(a) Representative mass histograms for wild-type yeast dyneinMOTOR with and without Pac1 with different nucleotides, as indicated (see Fig. 3 and Methods). Cartoon depictions above peaks indicate the likely dyneinMOTOR-Pac1 complex contained therein. (b) Histograms from mass photometry analysis depicting relative dyneinMOTOR-Pac1 complex formation in the presence of a fixed concentration of each motor (25 nM) and increasing concentrations of Pac1 (as indicated). Note the higher fraction of dyneinMOTORMT-U-Pac1 complex formation with respect to dyneinMOTORMT-B-Pac1 at every concentration. (c) Representative mass histograms for wild-type human dyneinMOTOR with and without LIS1 with different nucleotides, as indicated.

Extended Data Fig. 3 Vanadate-mediated photocleavage assay.

(a) Schematic depicting expected vanadate-mediated photocleavage if vanadate were bound to AAA1 (top) or AAA3 (bottom). (b and c) Two independent replicates of photocleavage assay. Purified indicated yeast dyneinMOTOR fragments were incubated with 3 mM ATP with or without 3 mM vanadate, exposed to ultraviolet light (254 nm) for 1 hour, and then analyzed by coomassie-stained SDS-PAGE.

Source data

Extended Data Fig. 4 Cryo-EM data processing flowchart.

(a) Cryo-EM workflow of MT-B in the presence of ATP (details in Methods). (b) FSC curves with the gold standard threshold of 0.143 for MT-B. (c, d) Particle distribution plot and 3D FSC analysis of MT-B. (e) Local resolution analysis of MT-B and representative cryo-EM densities. (f) Cryo-EM workflow of MT-U with LIS1. (g) FSC curves with the gold standard threshold of 0.143. (h, i) Particle distribution plot and 3D FSC analysis of MT-U + LIS1 in the presence of ATP and Vi. (j) Local resolution analysis and representative cryo-EM densities of the LIS1-LIS1 interface. (k) Cryo-EM workflow of MT-U alone in the presence of ATP and Vi. (l) FSC curves with the gold standard threshold of 0.143. (m, n) Particle distribution plot and 3D FSC analysis of MT-U. (o) Local resolution analysis and representative cryo-EM densities.

Extended Data Fig. 5 Additional analysis of human MT-U and MT-B cryo-EM structures.

(a) Stick representations of the dyneinMOTORMT-B AAA sites showing the nucleotide electron density (the center of each image) and surrounding residues (residues are color-coded as shown in panel b schematic). (b) Vector maps depicting pairwise alpha carbon interatomic distances between the dyneinMOTORMT-B with the following: apo yeast dynein (4AKG), AMPPNP-bound yeast dynein (4W8F), ADP-bound Dictyostelium discoideum dynein (3VKG), and ADP-Vi and Pac1-bound yeast dynein (7MGM)37,40,79,81. Structures were globally aligned after removal of the linkers. (c) AAA1-AAA2L domains from dyneinMOTORMT-B (shades of green) and the native microtubule-bound dynein-1 (7Z8G; magenta and pink) overlaid to depict the high degree of structural similarity. The two were locally aligned using AAA1. (d) AAA3-AAA4L domains from dyneinMOTORMT-B (shades of green) overlaid with either the native microtubule-bound dynein-1 (left, magenta and pink) or the ADP-bound Dictyostelium discoideum dynein (right, blue and green). (e and f) Stick representations of the LIS1-unbound (e) or bound (f) dyneinMOTORMT-U AAA sites showing the nucleotide electron density and surrounding residues (residues are color-coded as shown in panel b schematic). (g) Vector maps depicting pairwise alpha carbon interatomic distances between the LIS1-bound dyneinMOTORMT-U with those described for panel b. Structures were globally aligned after removal of the linkers. (h) AAA1-AAA2L domains from dyneinMOTORMT-U (left) and dyneinMOTORMT-B (right) with residues of dyneinMOTORMT-U contacting the Vi highlighted (E1959, Walker B; N2019, Sensor-I; R2358, arginine finger; N2316; A2354;). Distances between these residues and the Vi (or, for the dyneinMOTORMT-B structure, between them and where the Vi would be) are indicated.

Extended Data Fig. 6 Additional analysis of LIS1-bound dyneinMOTORMT-U structure.

(a) 2 LIS1-bound dyneinMOTORMT-U structure with domains colored as shown in Fig. 5 (left) and the same shown with the full-length LIS1 homodimer, with the N-terminal dimerization domain modeled. The LIS1 N-terminal dimer model was generated using a combination of AlphaFold prediction69 and a previous crystal structure, 1UUJ82. The structure was manually adjusted in COOT. (b) View of LIS1 homodimer surface that contacts sitestalk (teal) and sitering (cyan). Residues listed and indicated in different colors on the structure are those that make direct contact with dynein or LIS1. Residues with ‘*’ are those identified in a recent study to make contact with wild-type human dynein41. (c) Side-view of full-length LIS1 homodimer model with residues colored as in panel b. (d) Results of molecular dynamics simulation from Fig. 5g depicting change in interatomic distances in residues mediating contacts between LIS1 and dynein as a consequence of indicated mutations.

Extended Data Fig. 7 Sequence alignment of Pac1 and LIS1-binding regions within dynein.

Numbering corresponds to yeast dynein (Dyn1) sequence. Secondary structure indicated with cartoon helices (for alpha-helices) and arrows (for beta-strands).

Extended Data Fig. 8 Sequence alignment of dynein-binding regions within LIS1 and homologs.

Numbering corresponds to yeast Pac1 sequence. Secondary structure indicated with cartoon helices (for alpha-helices) and arrows (for beta-strands).

Extended Data Fig. 9 3D classification analysis of LIS1-bound dyneinMOTORMT-U structures.

(a) The three classes of LIS1-bound dynein (shown with a rotated close-up view of LIS1ring) are shown with local resolution indicated by color. Note the significant increase in resolution and map quality for LIS1ring for the ‘1.5’ and 2 LIS1-bound dyneins. (b) Close-up views of the indicated regions of the LIS1-bound dyneinMOTORMT-U structure. Note all three structures have clear density for ADP at AAA3 (top), and there is an improvement in local resolution for the AAA4 loop (middle) and the buttress for the ’1.5’ and 2 LIS1-bound structures. EM densities of each 3D class are shown along with models of dyneinMOTORMT-U and dyneinMOTORMT-U + 2 LIS1 for comparison in bottom two rows.

Extended Data Fig. 10 3D classification reveals flexibility of MT-U linker that is independent of LIS1 binding.

(a) 3D classification results for MT-U alone. Front views of the motor, and zoomed-in views of the linker (colored in purple) are shown. The linker position is indicated in the cartoon model. The map contour levels are all set to 0.45. Note that no obvious bent linker was observed in any class. (b) 3D classification results of MT-U with LIS1. The classification is mainly guided by LIS1 occupancy, but the linker flexibility can also be visualized at low map contour levels (all set to 0.2). For all classes, the linker position is flexible. No obvious bent linker was observed regardless of LIS1 occupancy.

Supplementary information

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Supplementary Results, Supplementary Discussion, Supplementary Figure 1, and Supplementary Table 1.

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Supplementary Video 1

Examples of Bik1-labeled astral microtubule plus ends statically associated with the cell cortex. Timelapse fluorescence images of cells expressing Bik1-3mCherry, GFP-Tub1, and Dyn1MT-U-3YFP (arrows in first frame of merge, astral microtubule plus ends with Bik1 foci statically associated with the cortex). Cartoons represent cells in first frame of movie (small green circle, spindle pole body; green lines, astral microtubules; magenta circles, plus end Bik1 foci statically associated with cortex).

Supplementary Video 2

Close-up views of dyneinMT-U-LIS1 contacts. Intermolecular contacts between dynein and LIS1, and between LIS1 and LIS1, are indicated with dashed lines. Nucleotide densities in AAA1 and AAA3 are indicated with mesh outlines.

Supplementary Video 3

Conformational differences within the AAA+ ring between MT-U and MT-B dynein. Structures for dyneinMOTORMT-U (+ LIS1) and dyneinMOTORMT-B were globally aligned (after removal of the linker and C-terminal domains). ChimeraX was used to morph between these two structures. Inset highlights the changes in AAA4-6 that appear to be initiated by the bending of the buttress. Note the rigid body reorientation of AAA5S-AAA6L, which transmits structural rearrangements to AAA6S and AAA1.

Supplementary Video 4

Conformational differences at the LIS1-binding sites between MT-U and MT-B dynein. Structures for dyneinMOTORMT-U (+ LIS1) and dyneinMOTORMT-B were locally aligned using AAA4-AAA5. ChimeraX was used to morph between these two structures. Shown are close-up views of sitestalk (left) and sitering (right). Residues with sidechains shown on LIS1 and dynein are those that mediate intermolecular contacts.

Supplementary Video 5

Model for Pac1/LIS1 function in budding yeast. See text for detailed description of model.

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Ton, W.D., Wang, Y., Chai, P. et al. Microtubule-binding-induced allostery triggers LIS1 dissociation from dynein prior to cargo transport. Nat Struct Mol Biol 30, 1365–1379 (2023). https://doi.org/10.1038/s41594-023-01010-x

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