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Structural studies on the stalk region of the axonemal and cytoplasmic dynein heavy chain

Ko, Seolmin 大阪大学 DOI:10.18910/93027

2023.09.25

概要

Title

Structural studies on the stalk region of the
axonemal and cytoplasmic dynein heavy chain

Author(s)

Ko, Seolmin

Citation

大阪大学, 2023, 博士論文

Version Type VoR
URL

https://doi.org/10.18910/93027

rights
Note

Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University

Form ３

Abstract of Thesis
Name

Title

（ Ko Seolmin）

Structural studies on the stalk region of the axonemal and cytoplasmic dynein heavy chain （軸
糸および細胞質ダイニン重鎖にあるストーク領域の構造研究）
Dynein motors, including axonemal and cytoplasmic dyneins, are an ATP-dependent microtubular motor

protein responsible for various biological processes and their dysfunction can lead to various diseases.
Despite its biological importance, structure-based dynein motor mechanisms remain unclear. In this study,
I focused on two specific aspects: the structural characterization of axonemal dynein stalk and the interaction
between cytoplasmic dynein microtubule-binding domain (MTBD) and microtubules.
For axonemal dynein, the X-ray crystal structure of human inner-arm dynein-d (DNAH1) with a long
coiled-coil stalk region was successfully determined as the first high resolution structure of axonemal dynein
stalk, enabling for a direct comparison with cytoplasmic and outer-arm dynein structures. The structural
comparison revealed distinctive characteristics in helical interaction, domain arrangement and flap motif
extension. Notably, additional analysis of knobs-into-hole packing of long coiled coil and relative domain
arrangement suggested a type-specific packing tightness and interacting pattern. Based on these findings,
a novel and isoform-specific "spike shoe model" was proposed to elucidate the role of the uniquely extended
flap motif in axonemal dynein. These findings provide valuable insights into the underlying mechanism of
axonemal dynein activity. Moreover, the first atomic structure of DNAH1 could be further used for a
representative example for future high-resolution axonemal inner-arm dynein complex studies.
Regarding cytoplasmic dynein, I aimed to investigate the structural details of the MTBD and its
interaction with microtubules. Previous attempts were limited by low-resolution structures or proteolysis
of the MTBD during crystallization. To overcome these challenges, I developed a new construct, modified
SRS-dynein fusion protein with α registry, fused with seryl tRNA-synthetase (SRS), to stabilize its coiled
coil registry to “alpha” with high binding affinity to microtubule. Additionally, I utilized the porcine

tubulin and D1 DARPin protein to form a triple complex of SRS-dynein, D1 DARPin, and the porcine tubulin dimer
for crystallization. Although I encountered difficulties with protein stability, I successfully obtained both
the triple complexed proteins and modified SRS-dynein crystals in the desired form. Finally, I propose using
she1 in future strategies to improve protein stability and gain a better understanding of the dynein-tubulin
interactions.
Overall, this study contributes to our understanding of axonemal and cytoplasmic dynein motors by providing
new insights into their structural characteristics and how they interact with microtubules.

様式 7

論文審査の結果の要旨及び担当者
氏

論文審査担当者

名

（

SEOLMIN

KO

）

（職）

氏

名

主査

教

授

栗栖源嗣

副査

教

授

原田慶恵

副査

教

授

中川敦史

副査

教

授

昆

隆英

論文審査の結果の要旨
学位申請者は，細胞内ではたらく分子モーターであるダイニンに着目し「Structural studies on the stalk
region of the axonemal and cytoplasmic dynein heavy chain（軸糸および細胞質ダイニン重鎖にあるストーク
領域の構造研究）」と題する研究を行った。
ダイニンは，AAA+スーパーファミリーに属する巨大な分子複合体で，ATP 加水分解のエネルギーをつかって力
発生し，微小管上をマイナス端方向へ移動するモーター分子である。生理的な機能に基づいて，ダイニンは軸糸
ダイニンと細胞質ダイニンに分類されている。細胞質ダイニンは，有糸分裂中の積荷輸送，細胞内輸送，鞭毛内
輸送を担っており，軸糸ダイニンは，周期的に配置された複数種のダイニン分子が活性化および不活性化を繰り
返すことで繊毛または鞭毛の波打ち運動を駆動する。軸糸ダイニンの機能不全は，原発性線毛運動不全症や精子
運動不全などの疾患を引き起こすことが知られている。このような生物学的重要性にもかかわらず，軸糸ダイニ
ンに存在する３種の外腕ダイニン（OADα，β，γ）と８種ある内腕ダイニンのアイソフォーム間での構造的違い
や，それぞれがどのように微小管と相互作用するのか，その様式の違いについては不明なことが多く残されてい
た。本論文で申請者は，ヒト内腕ダイニン d（DNAH1）の長い逆平行コイルドコイルと微小管結合ドメイン（MTBD）
を含むストーク領域の X 線結晶構造を 2.7 Å 分解能で決定した。これは内腕ダイニンのコイルドコイルを含む領
域の最初の構造であり，内腕ダイニンのアイソフォームに特徴的な構造の違いについて議論している。申請者が
着目したのは，ストーク領域のコイルドコイルと MTBD との相対的な配置が細胞質ダイニンとは異なっていた点で
ある。さらにβヘアピン構造をとる MTBD から突き出た“フラップ”と呼ばれる領域も，部分構造がわかっている
他 2 種の内腕ダイニンとは異なった向きに突き出ており，内腕ダイニン(DNAH1)と微小管の間の相互作用につい
て，アイソフォームごとに相互作用様式が異なるとする「スパイクシューモデル」を提案していることは特に注
目すべき点である。
さらに，ダイニンのコイルドコイルを安定化するセリル tRNA 合成酵素との融合タンパク質を設計し，アダプ
タータンパク質（DARPin)を加えて混合することで，微小管を構成するα，β-チューブリンとの三重複合体を形
成させて，安定な複合体が得られる方法を確立している。今後のダイニンと微小管との複合体構造研究につなが
る道を拓いた重要な成果である。
申請者は，軸糸ダイニンのうち構造情報が少なかった内腕ダイニンに着目し，DNAH1 のコイルドコイルと MTBD
との相対配置の違いが示唆するアイソフォーム特異的な微小管との相互作用モデルを提唱した。特にフラップ領
域の構造的な違いについて考察を行い，最終的に内腕ダイニンの微小管上でのステッピングについて新たなモデ
ルを提唱するに至っている。本論文の研究内容は，軸糸ダイニンの運動制御機構を理解する上で，大変意義のあ
る成果である。
よって，本論文は博士（理学）の学位論文として十分価値あるものと認める。

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List of publication

1. Seolmin Ko, Akiyuki Toda, Hideaki Tanaka, Jian Yu, Genji Kurisu (2023) Crystal structure

of the stalk region of axonemal inner-arm dynein-d reveals unique features in the coiled-coil

and microtubule-binding domain. FEBS Letters, available online ahead of print. Available from:

https://doi.org/10.1002/1873-3468.14690

...

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分野

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学位論文種類・取得年

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Structural studies on the stalk region of the axonemal and cytoplasmic dynein heavy chain

概要

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関連論文

The regulatory mechanisms and functional roles of the CENP-T-KMN network interaction for faithful chromosome segregation

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脂質モデル膜におけるガングリオシドGM3とEGF受容体膜貫通ペプチドの相互作用

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参考文献

分野

大学

学位論文種類・取得年

言語

コピーが完了しました

URLをコピーしました

Structural studies on the stalk region of the axonemal and cytoplasmic dynein heavy chain

概要

この論文で使われている画像

関連論文

The regulatory mechanisms and functional roles of the CENP-T-KMN network interaction for faithful chromosome segregation

A novel pathway of DNA double-strand break formation that is independent of loop-axis tethering

脂質モデル膜におけるガングリオシドGM3とEGF受容体膜貫通ペプチドの相互作用

Analysis of protein translocation across the chloroplast inner envelope membrane

Dynamic change of the CENP-C-centromere interaction is regulated by CDK1

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