2022
Taguchi, Shinya; Nakano, Juri; Imasaki, Tsuyoshi; Kita, Tomoki; Saijo-Hamano, Yumiko; Sakai, Naoki; Shigematsu, Hideki; Okuma, Hiromichi; Shimizu, Takahiro; Nitta, Eriko; Kikkawa, Satoshi; Mizobuchi, Satoshi; Niwa, Shinsuke; Nitta, Ryo
Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 –tubulin complex Journal Article
In: bioRxiv, 2022.
Abstract | Links | BibTeX | タグ:
@article{Taguchi2022.02.14.480441,
title = {Structural model of microtubule dynamics inhibition by Kinesin-4 from the crystal structure of KLP-12 –tubulin complex},
author = {Shinya Taguchi and Juri Nakano and Tsuyoshi Imasaki and Tomoki Kita and Yumiko Saijo-Hamano and Naoki Sakai and Hideki Shigematsu and Hiromichi Okuma and Takahiro Shimizu and Eriko Nitta and Satoshi Kikkawa and Satoshi Mizobuchi and Shinsuke Niwa and Ryo Nitta},
url = {https://www.biorxiv.org/content/early/2022/02/15/2022.02.14.480441},
doi = {10.1101/2022.02.14.480441},
year = {2022},
date = {2022-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Kinesin superfamily proteins are microtubule-based molecular motors driven by the energy of ATP hydrolysis. Among them, the kinesin-4 family is a unique motor that inhibits microtubule dynamics. Although mutations of kinesin-4 cause several diseases, its molecular mechanism is unclear because of the difficulty of visualizing the high-resolution structure of kinesin-4 working at the microtubule plus-end. Here, we report that KLP-12, a C. elegans kinesin-4 ortholog of KIF21A and KIF21B, is essential for proper length control of C. elegans axons, and its motor domain represses microtubule polymerization in vitro. The crystal structure of the KLP-12 motor domain complexed with tubulin, which represents the high-resolution structural snapshot of inhibition state of microtubule-end dynamics, revealed the bending effect of KLP-12 for tubulin. Comparison with the KIF5B-tubulin and KIF2C-tubulin complexes, which represent the elongation and shrinking forms of microtubule ends, respectively, showed the curvature of tubulin introduced by KLP-12 is in between them. Taken together, KLP-12 controls the proper length of axons by modulating the curvature of the microtubule ends to inhibit the microtubule dynamics.Competing Interest StatementThe authors have declared no competing interest.
Okuma, Hiromichi; Saijo-Hamano, Yumiko; Sherif, Aalaa Alrahman; Hoshizaki, Emi; Sakai, Naoki; Kato, Takaaki; Imasaki, Tsuyoshi; Nitta, Eriko; Sasai, Miwa; Maniwa, Yoshimasa; Kosako, Hidetaka; Standley, Daron M; Yamamoto, Masahiro; Nitta, Ryo
Structural basis of Irgb6 inactivation by~Toxoplasma gondii~through the phosphorylation of switch I. Journal Article
In: bioRxiv, 2022.
Abstract | Links | BibTeX | タグ:
@article{Okuma2022.10.31.514472,
title = {Structural basis of Irgb6 inactivation by~Toxoplasma gondii~through the phosphorylation of switch I.},
author = {Hiromichi Okuma and Yumiko Saijo-Hamano and Aalaa Alrahman Sherif and Emi Hoshizaki and Naoki Sakai and Takaaki Kato and Tsuyoshi Imasaki and Eriko Nitta and Miwa Sasai and Yoshimasa Maniwa and Hidetaka Kosako and Daron M Standley and Masahiro Yamamoto and Ryo Nitta},
url = {https://www.biorxiv.org/content/early/2022/11/01/2022.10.31.514472},
doi = {10.1101/2022.10.31.514472},
year = {2022},
date = {2022-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Upon infection with~Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite.~T. gondii~counters this response by releasing ROP18 kinase,~which inactivates IRG GTPases and inhibits their recruitment to the~T. gondii~parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has~lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from~the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding.~Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for~T. gondii~escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Upon infection with~Toxoplasma gondii, host cells produce immune-related GTPases (IRGs) to kill the parasite.~T. gondii~counters this response by releasing ROP18 kinase,~which inactivates IRG GTPases and inhibits their recruitment to the~T. gondii~parasitophorous vacuole (PV). However, the molecular mechanisms of this process are entirely unknown. Here we report the atomic structures of Irgb6 with a phosphomimetic mutation by ROP18. The mutant has~lower GTPase activity and is not recruited to the PV membrane (PVM). The crystal structure shows the mutant exhibit a distinct conformation from~the physiological nucleotide-free form, thus preventing GTPase cycling. This change allosterically modifies the conformation of the membrane-binding interface, preventing physiological PVM-binding.~Docking simulation of PI5P also supports the impaired binding of the mutant to PVM. We thus demonstrate the structural basis for~T. gondii~escape from host cell-autonomous defense, and provide a structural model for regulating enzymatic activity by phosphorylation.Competing Interest StatementThe authors have declared no competing interest.
2021
Imasaki, Tsuyoshi; Kikkawa, Satoshi; Niwa, Shinsuke; Saijo-Hamano, Yumiko; Shigematsu, Hideki; Aoyama, Kazuhiro; Mitsuoka, Kaoru; Aoki, Mari; Sakamoto, Ayako; Tomabechi, Yuri; Sakai, Naoki; Shirouzu, Mikako; Taguchi, Shinya; Yamagishi, Yosuke; Setsu, Tomiyoshi; Sakihama, Yoshiaki; Shimizu, Takahiro; Nitta, Eriko; Takeichi, Masatoshi; Nitta, Ryo
CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre Journal Article
In: bioRxiv, 2021.
Abstract | Links | BibTeX | タグ:
@article{Imasaki2021.03.01.433304,
title = {CAMSAP2 organizes a γ-tubulin-independent microtubule nucleation centre},
author = {Tsuyoshi Imasaki and Satoshi Kikkawa and Shinsuke Niwa and Yumiko Saijo-Hamano and Hideki Shigematsu and Kazuhiro Aoyama and Kaoru Mitsuoka and Mari Aoki and Ayako Sakamoto and Yuri Tomabechi and Naoki Sakai and Mikako Shirouzu and Shinya Taguchi and Yosuke Yamagishi and Tomiyoshi Setsu and Yoshiaki Sakihama and Takahiro Shimizu and Eriko Nitta and Masatoshi Takeichi and Ryo Nitta},
url = {https://www.biorxiv.org/content/early/2021/03/01/2021.03.01.433304},
doi = {10.1101/2021.03.01.433304},
year = {2021},
date = {2021-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, detachment of a considerable number of microtubules from the centrosome is known to contribute to fundamental processes in cells. Here, we present evidence that minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation from soluble αβ-tubulin independent of γ-tubulin. CAMSAP2 significantly reduces the nucleation barrier close to the critical concentration for microtubule polymerization by stabilizing the longitudinal contacts among αβ-tubulins. CAMSAP2 clusters together with αβ-tubulin to generate nucleation intermediates, from which numerous microtubules radiate, forming aster-like structures. Our findings suggest that CAMSAP2 supports microtubule growth by organizing a nucleation centre as well as by stabilizing microtubule nucleation intermediates.Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Microtubules are dynamic polymers consisting of αβ-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αβ-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, detachment of a considerable number of microtubules from the centrosome is known to contribute to fundamental processes in cells. Here, we present evidence that minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation from soluble αβ-tubulin independent of γ-tubulin. CAMSAP2 significantly reduces the nucleation barrier close to the critical concentration for microtubule polymerization by stabilizing the longitudinal contacts among αβ-tubulins. CAMSAP2 clusters together with αβ-tubulin to generate nucleation intermediates, from which numerous microtubules radiate, forming aster-like structures. Our findings suggest that CAMSAP2 supports microtubule growth by organizing a nucleation centre as well as by stabilizing microtubule nucleation intermediates.Competing Interest StatementThe authors have declared no competing interest.
Hamano, Yumiko Saijo; Sherif, Aalaa Alrahman; Pradipta, Ariel; Sasai, Miwa; Sakai, Naoki; Sakihama, Yoshiaki; Yamamoto, Masahiro; Standley, Daron M; Nitta, Ryo
Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6 Journal Article
In: bioRxiv, 2021.
Abstract | Links | BibTeX | タグ:
@article{Hamano2021.07.01.450801,
title = {Structural basis of membrane recognition of Toxoplasma gondii vacuole by Irgb6},
author = {Yumiko Saijo Hamano and Aalaa Alrahman Sherif and Ariel Pradipta and Miwa Sasai and Naoki Sakai and Yoshiaki Sakihama and Masahiro Yamamoto and Daron M Standley and Ryo Nitta},
url = {https://www.biorxiv.org/content/early/2021/07/01/2021.07.01.450801},
doi = {10.1101/2021.07.01.450801},
year = {2021},
date = {2021-01-01},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
abstract = {The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.Competing Interest StatementThe authors have declared no competing interest.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The p47 immunity-related GTPase (IRG) Irgb6 plays a pioneering role in host defense against Toxoplasma gondii infection. It is recruited to the parasitophorous vacuole membrane (PVM) formed by T. gondii and disrupts it. Despite the importance of this process, the molecular mechanisms accounting for PVM recognition by Irgb6 remain elusive due to lack of structural information on Irgb6. Here we report the crystal structures of mouse Irgb6 in the GTP-bound and nucleotide-free forms. Irgb6 exhibits a similar overall architecture to other IRGs in which GTP-binding induces conformational changes in both the dimerization interface and the membrane-binding interface. The membrane-binding interface of Irgb6 assumes a unique conformation, composed of N- and C-terminal helical regions forming a phospholipid binding site. In silico docking of phospholipids further revealed membrane binding residues that were validated through mutagenesis and cell-based assays. Collectively, these data demonstrate a novel structural basis for Irgb6 to recognize T. gondii PVM in a manner distinct from other IRGs.Competing Interest StatementThe authors have declared no competing interest.