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The mechanochemical cycle of reactive full-length human dynein 1

Nature Structural & Molecular Biology volume 32, pages 1383–1395 (2025)Cite this article

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Abstract

Dynein-driven cargo transport has a pivotal role in diverse cellular activities, central to which is dynein’s mechanochemical cycle. Here, we performed a systematic cryo-electron microscopic investigation of the conformational landscape of full-length human dynein 1 in reaction, in various nucleotide conditions, on and off microtubules. Our approach reveals over 40 high-resolution structures, categorized into eight states, providing a dynamic and comprehensive view of dynein throughout its mechanochemical cycle. The described intermediate states reveal mechanistic insights into dynein function, including a ‘backdoor’ phosphate release model that coordinates linker straightening, how microtubule binding enhances adenosine triphosphatase activity through a two-way communication mechanism and the crosstalk mechanism between AAA1 and the regulatory AAA3 site. Our findings also lead to a revised model for the force-generating powerstroke and reveal means by which dynein exhibits unidirectional stepping. These results improve our understanding of dynein and provide a more complete model of its mechanochemical cycle.

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Fig. 1: Cryo-EM analysis of full-length human dynein 1 off and on MTs.
Fig. 2: The conformational landscape of MT-unbound dynein motor domains in reaction.
Fig. 3: The release of Pi from AAA1 through a molecular backdoor mechanism.
Fig. 4: The mechanism for two-way communication between AAA1 and the MTBD.
Fig. 5: The mechanism for ATPase stimulation by MTs and dynein’s unidirectional stepping along MTs.
Fig. 6: Model for the mechanochemical cycle of full-length human dynein 1.

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Data availability

Models and cryo-EM maps were deposited to the PDB and EM Data Bank as outlined below.

Dynein in ATP buffer: PDB 9BLY and EMD-44681 (phi particle dynein), PDB 9BLZ and EMD-44682 (state 1), PDB 9BM0 and EMD-44683 (state 2), PDB 9BM1 and EMD-44684 (state 3), PDB 9BM2 and EMD-44685 (state 4), PDB 9BM3 and EMD-44686 (state 5a), PDB 9BM4 and EMD-44687 (state 5b), PDB 9BM5 and EMD-44688 (state 6), PDB 9BM6 and EMD-44689 (state 7a, post 2), PDB 9BM7 and EMD-44690 (state 7b, post 2) and PDB 9BM8 and EMD-44691 (state 7c, post 2).

Dynein in ATP-Vi buffer: PDB 9BMF and EMD-44697 (state 1), PDB 9BMG and EMD-44698 (state 2) and PDB 9BMH and EMD-44699 (state 4).

Dynein in 5 mM AMPPNP buffer with 2 mM Mg2+: PDB 9BMJ and EMD-44701 (state 1), PDB 9BML and EMD-44702 (state 2), PDB 9BMM and EMD-44703 (state 4), PDB 9BMN and EMD-44704 (state 5), PDB 9BMO and EMD-44705 (state 6) and PDB 9BMP and EMD-44706 (state 7, post 2).

Dynein in 5 mM AMPPNP buffer with 5 mM Mg2+: PDB 9DH5 and EMD-46856 (state 1), PDB 9DH6 and EMD-46857 (state 2), PDB 9DH7 and EMD-46858 (state 4), PDB 9DH8 and EMD-46859 (state 5), PDB 9DH9 and EMD-46860 (state 7, post 2) and PDB 9DHA and EMD-46861 (state 7, post 1)

Dynein in ADP buffer: PDB 9BMR and EMD-44708 (state 1), PDB 9BMS and EMD-44709 (state 2), PDB 9BMT and EMD-44710 (state 5), PDB 9BMU and EMD-44711 (state 6), PDB 9BMV and EMD-44712 (state 7, post 1) and PDB 9BMW and EMD-44714 (state 7, post 2).

Dynein in apo buffer: PDB 9BMY and EMD-44715 (state 1), PDB 9BMZ and EMD-44716 (state 2), PDB 9BN0 and EMD-44717 (state 7, post 2) and PDB 9BN1 and EMD-44718 (state 8, post 1).

Dynein bound to MTs: PDB 9BMA and EMD-44693 (apo, post 1), PDB 9BMB and EMD-44694 (ADP, post 1), PDB 9BMC and EMD-44695 (ADP, post 2) and PDB 9BMD and EMD-44696 (AMPPNP, post 1).

Dynein motor domain α registry mutant in ATP buffer: PDB 9BN3 and EMD-44720 (class 1) and PDB 9BN4 and EMD-44721 (class 2).

Dynein motor domain α registry mutant in ATP-Vi buffer: PDB 9BN5 and EMD-44722 (class 1) and PDB 9BN6 and EMD-44723 (class 2).

Previously reported atomic models used in this study (for model building and structural analysis) were obtained from the PDB under accession codes 5NUG, 7K58, 7K5B, 8FD6 and 7MGM. Additional data and materials can be obtained from the corresponding authors upon request. Source data are provided with this paper.

Code availability

All codes involved in general cryo-EM data processing and MT signal subtraction are publicly available from GitHub (https://github.com/JackZhang-Lab).

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Acknowledgements

We are very grateful to members of the K.Z. and S.M.M. laboratories for their valuable discussions. We thank R. Yang for the initial dynein 1 expression and preparations in the lab. This work was funded by the National Institutes of Health (NIH) National Institute of General Medical Sciences (R35GM139483 to S.M.M. and R35GM142959 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). We would like to thank K. Zhou, J. Lin, M. Llaguno and S. Wu, for their help with cryo-EM data collection at the Yale Cryo-EM facility. The Yale Cryo-EM resource is funded in part by the NIH grant S10OD023603 awarded to F. Sigworth. We thank L. Wang, J. Kaminsky and G. Hu at the LBMS for help with cryo-EM data collection. The LBMS is supported by the Department of Energy Office of Biological and Environmental Research (KP1607011).

Author information

Author notes

  1. Yue Wang

    Present address: Innovation Center for Brain Medical Sciences, The Ministry of Education of the People’s Republic of China, Huazhong University of Science and Technology, Wuhan, China

  2. These authors contributed equally: Pengxin Chai, Jun Yang.

  3. These authors jointly supervised this work: Steven M. Markus, Yue Wang, Kai Zhang

Authors and Affiliations

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

    Pengxin Chai, Jun Yang, Yue Wang & Kai Zhang

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

    Indigo C. Geohring & Steven M. Markus

  3. Department of Pathophysiology, School of Basic Medicine, Huazhong University of Science and Technology, Wuhan, China

    Yue Wang

Authors
  1. Pengxin Chai
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  2. Jun Yang
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  3. Indigo C. Geohring
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  4. Steven M. Markus
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Contributions

K.Z. designed the study. J.Y. and Y.W. purified the full-length human proteins. J.Y., Y.W. and P.C. prepared the cryo-EM samples and collected the data. P.C., J.Y., Y.W. and K.Z. processed the images and built the PDB models. S.M.M. purified the human MT-B dynein motor domain. I.C.G. generated the yeast strains and purified the proteins from yeast. I.C.G. and S.M.M. performed and analyzed the ATPase and single-molecule assays. P.C., J.Y. Y.W., K.Z. and S.M.M. generated the figures and videos. P.C., J.Y. and K.Z. wrote the manuscript. P.C., J.Y., Y.W., K.Z. and S.M.M. edited and revised the manuscript. S.M.M. and K.Z. acquired funding.

Corresponding authors

Correspondence to Steven M. Markus, Yue Wang or Kai Zhang.

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Competing interests

The authors declare no competing interests.

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Peer review information

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

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Extended data

Extended Data Fig. 1 Cryo-EM data processing of full-length human dynein-1 in the presence of 5 mM ATP.

(a) A representative negative staining micrograph (total 100 micrographs) of purified full-length human dynein-1 and corresponding 2D class averages. (b) A typical cryo-EM micrograph (total 33,302 micrographs) and the flowchart of cryo-EM image processing. (c) Image processing of full-length phi dynein and tail region. (d, e) Orientation distribution of phi dynein and a representative state from open dynein. (f, g) The Fourier shell correlation (FSC) curves of the motor domain in different states and the tail domain. Datasets for dynein in other nucleotide conditions (ATP-Vi, AMPPNP, ADP, apo) were processed similarly.

Extended Data Fig. 2 Local resolution analysis and identification of bound nucleotides in AAA1, AAA3, and AAA4 in different states from the dynein-ATP dataset.

The color scheme for the motor domain is consistent with Fig. 4.

Extended Data Fig. 3 Cryo-EM data processing of full-length human dynein-1 bound to MTs.

(a) Flowchart of sample preparation and typical cryo-EM micrographs of dynein bound to MTs in apo, ADP, and AMPPNP conditions. The number of micrographs is shown in the panel. (b) Microtubule signal subtraction and image processing flowchart. (c) Orientation distribution of dynein-MT-ADP reconstruction. (d) FSC curves of four MT-bound motors. (e) Plot for the full-length dynein bound to MTs in different conformations (two stable heads, one stable trailing head, and one stable leading head).

Extended Data Fig. 4 Cryo-EM analysis of dynein in ATP-Vi and AMPPNP conditions.

(a) Cartoon schematic depicting the experimental method for cryo-EM analysis of full-length dynein in different nucleotide conditions. (b) Typical cryo-EM micrographs of dynein in different nucleotide conditions, with total number of micrographs shown in the panel. (c, d, e) FSC curves and local resolution analysis of dynein in ATP-Vi, AMPPNP-low Mg2+, and AMPPNP-high Mg2+ conditions.

Extended Data Fig. 5 Cryo-EM analysis of dynein in ADP and apo conditions and dynein-bound to microtubules.

(a, b) FSC curves and local resolution analysis of dynein in ADP and apo conditions. (c) FSC curves and local resolution analysis of dynein-bound to microtubules.

Extended Data Fig. 6 The conformational landscapes of active cycle motor domains in different nucleotide conditions.

(a-f) Conformation landscapes of motor domains in ATP, ATP-Vi, AMPPNP, ADP, and apo conditions. Arrows indicate mechanochemical pathways. Percentages indicate the proportion of particles in each state. The missing states in each condition are shown in grey. Phi dynein is not included in this figure.

Extended Data Fig. 7 Cryo-EM densities of the nucleotide and sensor-I loop in various closed AAA1 states.

(a) From this work. (b) From previously published structures (PDB:8FD6 ref. 21, 7MGM ref. 78). The conformations of N2019 and Y2022 are highlighted.

Extended Data Fig. 8 Structural comparisons of MT-bound and -unbound dynein motors.

(a) Comparison between MT-dynein in apo condition with state-8 motor domain. (b) Comparison between MT-dynein in ADP condition with state-7 motor domains. (c) Comparison between MT-dynein in AMPPNP condition with state-7 motor domains. The nucleotide states in AAA1, AAA3, and AAA4 and the linker docking mode are listed. The MT-bound dynein motor domains (from linker to C-terminus) were used as references for structural fitting in ChimeraX.

Extended Data Fig. 9 Systematic analysis of linker-ring interactions among four MT-bound motor domains.

(a) Cartoon and molecular models showing the interaction interfaces including linker-AAA2L, linker-AAA5L, and linker-AAA3-4-connector helix. (b-e) Comparison of linker docking modes between dynein-1 and outer-arm dynein.

Extended Data Fig. 10 Structural survey of motor domains and statistical analysis of crosstalk between AAA1 and AAA3.

(a) Flowchart of structural survey of 56 motor domain structures. (b) Statistical graphs showing how AAA1 communicates to AAA3, linker and MTBD. (c) Statistical graphs showing how AAA3 communicates to AAA1, linker and MTBD. (d) Cartoon model summarizing communication between AAA1, AAA3, linker and MTBD.

Supplementary information

Supplementary Information

Supplementary Notes 1–5, Figs. 1 and 2 and Tables 1–8.

Reporting Summary

Peer Review File

Supplementary Data 1

Features of the motor domains in different states.

Supplementary Data 2

Structural survey of the dynein 1 motor domains.

Supplementary Video 1

High-resolution cryo-EM map of phi dynein and representative local density maps.

Supplementary Video 2

High-resolution cryo-EM reconstruction of dynein bound to MTs.

Supplementary Video 3

The release of Pi from dynein AAA1 through the backdoor mechanism, triggering the linker straightening.

Supplementary Video 4

Communication between AAA1 pocket dynamics and MTBD.

Supplementary Video 5

The forward-stepping mechanism of dynein along MTs.

Source data

Source Data Fig. 1

Statistical source data (dynein conformational distribution).

Source Data Fig. 2

Statistical source data (ATPase values).

Source Data Fig. 3

Statistical source data (single-molecule motility data).

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Chai, P., Yang, J., Geohring, I.C. et al. The mechanochemical cycle of reactive full-length human dynein 1. Nat Struct Mol Biol 32, 1383–1395 (2025). https://doi.org/10.1038/s41594-025-01543-3

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