ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites

Imai, Hiroshi; Kanazawa, Riko; Maeshima, Toshihisa; Tran, Duy Phuoc; Shimo-Kon, Rieko; Tokita, Hisaya; Miyata, Tomoko; Makino, Fumiaki; Fujita, Junso; Kato, Takayuki; Kitao, Akio; Namba, Keiichi; Kon, Takahide

doi:https://doi.org/10.1016/j.jmb.2026.169977

ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites

Imai, Hiroshi; Kanazawa, Riko; Maeshima, Toshihisa et al.

Journal of Molecular Biology, 2026, 438(21), 169977

Number of Access:92（2026-10-11 15:24 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/106628

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Title	ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites
Creator	NRID 1000060391869 1000060391869 ORCID 0000-0001-8939-2255 0000-0001-8939-2255 researchmap permalink Hiroshi_Imai Hiroshi_Imai Scopus Author ID 55628551788 55628551788 CreatorName Imai, Hiroshi
	CreatorName Kanazawa, Riko
	CreatorName Maeshima, Toshihisa
	NRID 1000050848546 1000050848546 CreatorName Tran, Duy Phuoc
	ORCID 0009-0002-6610-0365 0009-0002-6610-0365 Scopus Author ID 24475899300 24475899300 CreatorName Shimo-Kon, Rieko
	CreatorName Tokita, Hisaya
	NRID 1000030423156 1000030423156 ORCID 0000-0002-5543-7459 0000-0002-5543-7459 researchmap permalink miya_t miya_t Scopus Author ID 7401764889 7401764889 CreatorName Miyata, Tomoko
	ORCID 0000-0001-9512-7087 0000-0001-9512-7087 Scopus Author ID 55604852800 55604852800 CreatorName Makino, Fumiaki
	NRID 1000080876368 1000080876368 CreatorName Fujita, Junso
	NRID 1000020423155 1000020423155 ORCID 0000-0002-8879-6685 0000-0002-8879-6685 researchmap permalink tkato_ipr tkato_ipr Scopus Author ID 55261010900 55261010900 CreatorName Kato, Takayuki
	NRID 1000030252422 1000030252422 CreatorName Kitao, Akio
	NRID 1000030346142 1000030346142 ORCID 0000-0003-2911-5875 0000-0003-2911-5875 researchmap permalink read0096830 read0096830 Scopus Author ID 7103162863 7103162863 CreatorName Namba, Keiichi
	NRID 1000030332620 1000030332620 ORCID 0000-0002-1231-7609 0000-0002-1231-7609 researchmap permalink read0067779 read0067779 Scopus Author ID 7005425389 7005425389 CreatorName Kon, Takahide
Subject	AAA+ATPase
	cryo-electron microscopy
	dynein
	molecular dynamics simulation
	motor protein
Description	Imai H., Kanazawa R., Maeshima T., et al. ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites. Journal of Molecular Biology 438, 169977 (2026); https://doi.org/10.1016/j.jmb.2026.169977.
Abstract	Cytoplasmic dynein is essential for intracellular transport and cell division, and its dysfunction is implicated in severe neurological disorders. Its motility is driven by the primary ATPase site in the AAA1 module and allosterically modulated by regulatory ATPase sites in the AAA3 and AAA4 modules. Despite recent structural advances, post-force-generating (post-power-stroke) transitions and the structural basis of AAA3/AAA4-mediated regulation remain elusive. Using cryo-electron microscopy, we captured intermediate structures of Dictyostelium discoideum cytoplasmic dynein. We identified two distinct ADP-bound states and two apo states, the latter comprising a partially nucleotide-free state with ADP retained at the AAA3 and AAA4 ATPase sites and a fully nucleotide-free state. For the AAA1 ATPase site, our structures and molecular dynamics simulations suggest a dynamic equilibrium between two ADP-bound states. In this model, linker swing and docking onto the AAA5 module can be driven by thermal fluctuations while ADP remains bound at the AAA1 ATPase site, shifting the motor from a canonical ADP-bound state to a metastable apo-like conformation with reduced ADP affinity. This transition could provide a framework for tension-sensing coordination. For the AAA3 and AAA4 ATPase sites, comparison of the partially and fully nucleotide-free apo structures indicates that ADP release induces localized outward tilting of the corresponding large submodules. We propose that this rearrangement displaces the AAA4 Pre-Sensor-I insert from the linker, thereby disfavoring the recovery stroke and stabilizing an inactive state. Together, our findings provide a mechanistic model in which coordinated conformational dynamics across multiple ATPase sites govern dynein’s chemomechanical cycle.
Publisher	Academic Press
Source Title	Journal of Molecular Biology
Volume (Issue)	438(21)
Page	169977
Date of Issued	2026-11-01
Language	English
Handle URL	https://hdl.handle.net/11094/106628
PISSN	00222836
eISSN	10898638
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1016/j.jmb.2026.169977
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution 4.0 International License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	Text
DCTERMS.bibliographicCitation	Journal of Molecular Biology. 2026, 438(21), p. 169977
DC.title	ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites
DC.creator	Imai, Hiroshi
	Kanazawa, Riko
	Maeshima, Toshihisa
	Tran, Duy Phuoc
	Shimo-Kon, Rieko
	Tokita, Hisaya
	Miyata, Tomoko
	Makino, Fumiaki
	Fujita, Junso
	Kato, Takayuki
	Kitao, Akio
	Namba, Keiichi
	Kon, Takahide
DC.publisher	Academic Press
DC.language' scheme='DCTERMS.RFC1766	English
DC.type' scheme='DCTERMS.DCMIType	Text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2026-11-01
DC.identifier	https://doi.org/10.1016/j.jmb.2026.169977
DC.subject	AAA+ATPase
	cryo-electron microscopy
	dynein
	molecular dynamics simulation
	motor protein
DC.description	Imai H., Kanazawa R., Maeshima T., et al. ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites. Journal of Molecular Biology 438, 169977 (2026); https://doi.org/10.1016/j.jmb.2026.169977.
DCTERMS.abstract	Cytoplasmic dynein is essential for intracellular transport and cell division, and its dysfunction is implicated in severe neurological disorders. Its motility is driven by the primary ATPase site in the AAA1 module and allosterically modulated by regulatory ATPase sites in the AAA3 and AAA4 modules. Despite recent structural advances, post-force-generating (post-power-stroke) transitions and the structural basis of AAA3/AAA4-mediated regulation remain elusive. Using cryo-electron microscopy, we captured intermediate structures of Dictyostelium discoideum cytoplasmic dynein. We identified two distinct ADP-bound states and two apo states, the latter comprising a partially nucleotide-free state with ADP retained at the AAA3 and AAA4 ATPase sites and a fully nucleotide-free state. For the AAA1 ATPase site, our structures and molecular dynamics simulations suggest a dynamic equilibrium between two ADP-bound states. In this model, linker swing and docking onto the AAA5 module can be driven by thermal fluctuations while ADP remains bound at the AAA1 ATPase site, shifting the motor from a canonical ADP-bound state to a metastable apo-like conformation with reduced ADP affinity. This transition could provide a framework for tension-sensing coordination. For the AAA3 and AAA4 ATPase sites, comparison of the partially and fully nucleotide-free apo structures indicates that ADP release induces localized outward tilting of the corresponding large submodules. We propose that this rearrangement displaces the AAA4 Pre-Sensor-I insert from the linker, thereby disfavoring the recovery stroke and stabilizing an inactive state. Together, our findings provide a mechanistic model in which coordinated conformational dynamics across multiple ATPase sites govern dynein’s chemomechanical cycle.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	106628
citation_title	ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites
citation_author	Imai, Hiroshi
	Kanazawa, Riko
	Maeshima, Toshihisa
	Tran, Duy Phuoc
	Shimo-Kon, Rieko
	Tokita, Hisaya
	Miyata, Tomoko
	Makino, Fumiaki
	Fujita, Junso
	Kato, Takayuki
	Kitao, Akio
	Namba, Keiichi
	Kon, Takahide
citation_publisher	Academic Press
citation_language	English
citation_date	2026-11-01
citation_journal_title	Journal of Molecular Biology
citation_volume	438
citation_issue	21
citation_firstpage	169977
citation_doi	https://doi.org/10.1016/j.jmb.2026.169977
citation_public_url	https://hdl.handle.net/11094/106628

ADP-Bound States of Cytoplasmic Dynein: Cryo-electron Microscopy Reveals a Two-Step Post-Power-Stroke Transition and Roles of Regulatory ATPase Sites

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