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Nde1 promotes Lis1 binding to full-length autoinhibited human dynein 1

Nature Chemical Biology (2025)Cite this article

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Abstract

Cytoplasmic dynein 1 (dynein) is the primary motor responsible for the retrograde transport of intracellular cargoes along microtubules. Activation of dynein requires the opening its autoinhibited Phi conformation, a process driven by Lis1 and Nde1/Ndel1. Using biochemical reconstitution and cryo-electron microscopy, we demonstrate that Nde1 enhances Lis1 binding to autoinhibited dynein and facilitates Phi opening. We identify a key intermediate in this activation pathway where a single Lis1 dimer binds between Phi-like (PhiL) motor rings. In this ‘PhiL–Lis1’ complex, Lis1 interacts with one motor domain through canonical sites at the AAA+ (adenosine triphosphatases associated with diverse cellular activities) ring and stalk, and with AAA5, AAA6 and linker regions of the other motor domain. Mutagenesis and motility assays confirm the critical role of the PhiL–Lis1 interface in dynein activation. This intermediate forms rapidly in the presence of Nde1, although Nde1 is not part of PhiL–Lis1. These findings provide key insights into how Nde1 promotes Lis1-mediated Phi opening.

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Fig. 1: Nde1 promotes Lis1 binding to Phi dynein and synergistically releases dynein autoinhibition.
Fig. 2: Structure of the PhiL–Lis1 complex.
Fig. 3: Lis1 induces a transition in the Phi conformation to accommodate its binding.
Fig. 4: Interactions identified between Lis1ring and MD-A in PhiL–Lis1.
Fig. 5: Comparison of the interface between MD-A and MD-B in Phi and PhiL–Lis1.
Fig. 6: Model for the initial stage of dynein activation by Nde1 and Lis1.

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Data availability

Cryo-EM density maps and models were deposited to the EM Data Bank and PDB under the following accession numbers: dynein and Lis1 condition, PDB 9E12 and EMD-47381 for full-length Phi, PDB 9E10 and EMD-47379 for the motor domains of Phi, PDB 9E13 and EMD-47382 for full-length PhiL–Lis1 and PDB 9E11 and EMD-47380 for motor domains of PhiL–Lis1; in the dynein, Lis1 and Nde1 condition, PDB 9E14 and EMD-47383 for full-length PhiL–Lis1 and PDB 9E0Z and EMD-47378 for motor domains of PhiL–Lis1. Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the K.Z. and A.Y. laboratories for their valuable discussions. This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (GM136414 to A.Y. and GM142959 to K.Z.) and in part by a Collaboration Development Award Program (to K.Z.) from the Pittsburgh Center for Human Immunodeficiency Virus Protein Interactions (U54AI170791). The cryo-EM data were collected at the Yale ScienceHill cryo-EM facility. We thank J. Lin and K. Zhou for assistance with the data collection. The Yale Cryo-EM Resource is funded in part by the NIH grant S10OD023603 awarded to F. J. Sigworth.

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Author notes

  1. These authors contributed equally: Jun Yang, Yuanchang Zhao, Pengxin Chai.

Authors and Affiliations

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

    Jun Yang, Pengxin Chai & Kai Zhang

  2. Physics Department, University of California, Berkeley, Berkeley, CA, USA

    Yuanchang Zhao & Ahmet Yildiz

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA

    Ahmet Yildiz

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  1. Jun Yang
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Contributions

K.Z. and A.Y. designed the study. J.Y. expressed and purified the dynein, Lis1 and Nde1 proteins for EM. J.Y. and P.C. prepared the cryo-EM samples, collected and processed the data and built the PDB models. P.C. and J.Y. processed the negative-stain EM data and quantified the particle numbers. Y.Z. performed the Lis1 mutagenesis, protein preparation, TIRF imaging and MP assays. J.Y., P.C., Y.Z., K.Z. and A.Y. analyzed the data and prepared the figures. J.Y., Y.Z., P.C., K.Z. and A.Y. wrote the paper.

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Correspondence to Ahmet Yildiz or Kai Zhang.

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Extended data

Extended Data Fig. 1 MP analysis of nucleotide conditions on Nde1 and Lis1 binding to WT dynein.

MP shows that under apo buffer (a), 0.1 mM ATP (b), ADP (c), ATP.vi (d), and AMPPNP (e) conditions, Nde1 promotes Lis1 binding to dynein, forming a 1:1 dynein-Lis1 (DL) complex. The nucleotide condition does not affect Nde1’s ability to tether Lis1 to dynein. Importantly, the formation of dynein-Nde1, dynein-Lis1-Nde1, and 1:2 dynein-Lis1 complexes was not observed. In the Lis1-alone condition, no significant DL complex was formed immediately.

Extended Data Fig. 2 Workflow for negative-stain EM data processing.

a, Representative micrographs for dynein alone (42 micrographs), dynein-Lis1 at 1:1 (41 micrographs), and dynein-Lis1 at 1:2 (47 micrographs) molar ratios from batch #1. b, Particle picking from representative micrographs in each dataset using a template matching approach based on Phi and open dynein models (particle diameter: 750 Å, distance cutoff: 400 Å). c, Three rounds of 2D classification were performed after extracting all particles (box size: 960 Å × 960 Å), yielding class averages of Phi dynein, open dynein motors, single motors, and junk particles. d, Final classified 2D averages showing Phi dynein (43.7%), two-motor open dynein (43.3%), and single-motor open dynein (13.0%). The particle numbers for each group were counted, and single motors were considered as open dynein by dividing the total number of particles by two.

Extended Data Fig. 3 Quantification of Phi-dynein percentage in the presence or absence of Lis1 and Nde1.

Dynein (D) was incubated with Lis1 (L), Nde1 (N), or both proteins for 90 minutes at the indicated molar ratios of protein dimers. The percentage of Phi-dynein was quantified under each condition, D + L (a), D + N (b), D + L + N (c). Colors represent independent biological replicates. n = 6 biological replicates for D:L = 1:1 and D:L = 1:2; n = 3 biological replicates for D:N = 1:1 and n = 4 biological replicates for D:N = 1:2; n = 4 biological replicates for D:L:N = 1:1:1 and D:L:N = 1:2:2. The control (Ctrl) corresponds to the Phi-dynein percentage measured in the dynein-alone condition.

Source data

Extended Data Fig. 4 Cryo-EM data processing for the dynein-Lis1 dataset.

a, A representative cryo-EM micrograph and the flowchart of cryo-EM data processing. b, Fourier Shell Correlation (FSC) curves showing the final resolution estimates for the motor domains of the Phi (2.71 Å) and PhiL-Lis1(2.86 Å) datasets. Orientation distribution of Phi (c) and PhiL-Lis1(d).

Extended Data Fig. 5 Cryo-EM data processing for the dynein-Lis1-Nde1 dataset.

a, A representative cryo-EM micrograph and the flowchart of cryo-EM data processing. b, FSC curve showing the final resolution estimate for the motor domains of the PhiL-Lis1 (2.88 Å) dataset. c, Comparison of Nde1’s effect on Lis1 binding to the open dynein motor. Cryo-EM particle numbers for open dynein-Lis1 and open dynein alone were quantified from the dynein-Lis1 and dynein-Lis1-Nde1 datasets. In the dynein-Lis1 dataset, 493,173 particles correspond to single dynein motors unbound to Lis1, and 624,475 particles correspond to dynein motors bound to Lis1 (including Lis1 dimers and monomers). In the dynein-Lis1-Nde1 dataset, 162,229 particles correspond to dynein motors unbound to Lis1, and 170,995 particles correspond to dynein motors bound to Lis1 (including Lis1 dimers and monomers), indicating that Nde1 does not promote Lis1 binding to the open dynein motor.

Extended Data Fig. 6 Comparison of local resolution, nucleotide binding in AAA1, AAA3, AAA4, and sensor-I loop conformation in MD-A of the Phi and PhiL-Lis1.

Local resolution, and nucleotide binding states in MD-A at AAA1, AAA3, and AAA4 of the Phi (a) and PhiL-Lis1 (b). MD-A and -B share the same nucleotide binding in AAA1, AAA3, and AAA4 across both the Phi and PhiL-Lis1. The sensor-I loop adopts almost the same conformation in MD-A (or -B) of both Phi (c) and PhiL-Lis1 (d), indicating that Lis1 binding does not affect phosphate release. The color scheme is the same as Fig. 4.

Extended Data Fig. 7 Density quality at the dynein MD-A and Lis1 interface in PhiL-Lis1.

a, Well-defined density at the AAA5-Lis1ring and AAA6-Lis1ring regions, showing tight and stable interactions. The color scheme for the motor domains is consistent with Fig. 4. b, Flexible density at the linker-Lis1ring interface, indicating dynamic interactions in this region.

Extended Data Fig. 8 Binding characterization of Lis1 mutants and structural prediction of Lis1Δ300–304.

a, MP profiles show binding of Lis1 mutants to open dynein. Open dynein and Lis1 mutants were mixed at a 1:2 ratio and incubated for 2 min prior to measurements. b, MP profiles show binding of the Lis1AAA6 mutant to dynein with Nde1 at different incubation times. Dynein, Lis1AAA6, and Nde1 were mixed at 1:2:2 ratio (D: dynein only, DL: one dynein and one Lis1, DL2N: one dynein, two Lis1s, and one Nde1) and incubated for 2, 6, and 15 minutes before the measurements. c, The percentages of mass populations detected in b. d, Overlay of AlphaFold3-predicted Lis1Δ300-304 and WT Lis1 bound to PhiL dynein motor domains. Lis1Δ300-304 is shown in violet, while WT Lis1 is colored green and sky blue. Dynein motors domains are displayed in grey and white. The structure overlay indicates that deletion of residues 300–304 in Lis1 surface loop does not induce notable conformational change of the rigid elements of Lis1Δ300-304.

Extended Data Fig. 9 Characterization of additional mutants of Lis1 at the PhiL-Lis1 interface.

a, MP profiles illustrate the interaction of Nde1 (N) with Lis1 mutants. Nde1 interacts with one (NL) or two (NL2) Lis1 dimers. b, MP of the binding of Lis1 mutants to open dynein. Dynein and Lis1 were incubated for 2 minutes at 1:2 ratio (D: dynein only, DL: one dynein and one Lis1). c. MP shows the binding of Lis1 mutants to dynein with or without Nde1. Dynein, Lis1 and Nde1 were incubated for 2 minutes at 1:2:2 ratio (DL2N: one dynein, two Lis1s, and one Nde1). d, Representative kymographs show the motility of WT DDR complexes with or without Nde1 and Lis1. e, Run frequency of WT DDR with or without Nde1 and Lis1 (mean ± s.d.; n = 30 MTs for each condition; statistics from two independent experiments). Results were normalized to the -Lis1, -Nde1 condition. P values are calculated from a two-tailed t-test.

Source data

Extended Data Fig. 10 Comparison of yeast Chi-Lis1 and human PhiL-Lis1 motor domains.

a, The structure of yeast Chi-Lis1 (PDB:8DZZ)41, showing two tail-truncated yeast dynein motor domains (grey) bound to two Lis1 dimers (colored, Chi-Lis1 1:2). b, Residues of MD-A that interact with Lis1ring are located in AAA6-Lis1ring and AAA5-Lis1ring regions and highlighted with dashed rectangle. Representative residues of MD-A involved in the canonical Lis1ring binding sites are located in Lis1ring-AAA3, Lis1ring-AAA4, Lis1ring-AAA5 and Lis1ring-stalk region. Interactions between MD-A and MD-B are in stalk-stalk region. Residues are displayed in sphere mode and are colored according to the subdomains in Fig. 4. c, Superimposition of the human PhiL-Lis1 and yeast Chi-Lis1 structures, showing that Chi-Lis1 adopts a more expanded conformation, with larger grooves on both the front and back sides compared to the more compact PhiL-Lis1 structure. Lis1 is hidden for clarity. Vectors represent interatomic distances of pairwise Cα atoms between the PhiL-Lis1 and Chi-Lis1 structures.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Tables 1–4.

Reporting Summary

Supplementary Video 1

Full-length human dynein in PhiL conformation, bound to a Lis1 dimer and displaying the newly identified interface with Lis1.

Supplementary Video 2

Single-molecule motility recordings of WT DDR complexes, in the presence or absence of Nde1, WT Lis1 and Lis1 mutants. The fluorescence signal originates from BicDR1–mNeonGreen.

Source data

Source Data Fig. 1

Statistical source data of relative percentage of Phi dynein.

Source Data Fig. 4

Statistical source data of dynein motility under Lis1 mutants.

Source Data Extended Data Fig. 3

Statistical source data of Phi dynein percentage.

Source Data Extended Data Fig. 9

Statistical source data of dynein motility under additional Lis1 mutants.

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Yang, J., Zhao, Y., Chai, P. et al. Nde1 promotes Lis1 binding to full-length autoinhibited human dynein 1. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-01981-6

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