Functions and Fine Structure of the AAA Protease FtsH

AAA蛋白酶FtsH的功能和精细结构

• 批准号: 11694222
• 负责人: OGURA Teru
• 金额: $ 1.98万
• 依托单位: KUMAMOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11694222/
• 关键词: ATPase; protease; chaperone; stress response; lambda phage; plasmid partition; sporulation; タンパク質複合体; コンピューターモデリング

We have studied structure and cellular functions of an essential, membrane-bound ATP-dependent protease, FtsH in E.coli. FtsH forms oligomers, and oligomerization is essential for its activity. Site-directed mutagenesis and homology modeling revealed that the ATP-binding pocket is formed at the interface of neighboring subunits, and that invariant residues in the AAA ATPase-specific sequence, SRH, play important roles in ATP hydrolysis. These results have led us to propose an intermolecular catalysis model for the hexameric AAA ATPase. The model also suggests that unfolded substrates are threaded through the central hole of the hexameric ring of the ATPase, and that the size of the central hole is altered upon ATP hydrolysis. We have succeeded to crystallize the ATPase domain of FtsH, however X-ray diffraction data have not been sufficient to resolve the crystal structure. FtsH controls several cellular functions by degrading specific regulatory proteins such as sigma32 and LpxC.Sigma32 is the heat shock transcription factor and is degraded by FtsH.Efficient degradation of sigma32 by FtsH requires the DnaK chaperone system, which may alter sigma32 to a form competent to the FtsH action. We isolated two temperature-sensitive ftsH mutants newly, and mutations of these mutants were mapped to the C-terminal region of ftsH.Mini-F plasmids are unstably maintained in temperature-sensitive ftsH mutants at permissive temperatures for cell growth. We have also studied lysis-lysogeny decision of lambda phage and colicin tolerance in ftsH mutants. We have identified SpoVM as a substrate of B.subtilis FtsH, suggesting Ftsh's involvement in sporulation. SpoVM acts for the membrane and exerts an inhibitory effect on cell growth.

我们研究了大肠杆菌中一种必需的膜结合 ATP 依赖性蛋白酶 FtsH 的结构和细胞功能。 FtsH 形成寡聚体，而寡聚化对其活性至关重要。定点诱变和同源建模揭示了 ATP 结合袋是在相邻亚基的界面处形成的，并且 AAA ATP 酶特异性序列 SRH 中的不变残基在 ATP 水解中发挥着重要作用。这些结果使我们提出了六聚体 AAA ATP 酶的分子间催化模型。该模型还表明，未折叠的底物穿过 ATP 酶六聚环的中心孔，并且中心孔的大小在 ATP 水解时发生改变。我们已经成功结晶了 FtsH 的 ATPase 结构域，但是 X 射线衍射数据不足以解析晶体结构。 FtsH 通过降解特定的调节蛋白（例如 sigma32 和 LpxC）来控制多种细胞功能。Sigma32 是热休克转录因子，可被 FtsH 降解。FtsH 有效降解 sigma32 需要 DnaK 伴侣系统，该系统可能会将 sigma32 改变为能够胜任的形式。 FtsH 行动。我们新分离出了两个温度敏感的ftsH突变体，这些突变体的突变被定位到ftsH的C端区域。Mini-F质粒在温度敏感的ftsH突变体中在细胞生长允许的温度下不稳定地维持。我们还研究了 ftsH 突变体中 lambda 噬菌体的裂解溶原决定和大肠菌素耐受性。我们已确定 SpoVM 是枯草芽孢杆菌 FtsH 的底物，表明 Ftsh 参与孢子形成。 SpoVM 作用于细胞膜，对细胞生长发挥抑制作用。

项目成果

Karata,K.,Verma,C.S.,Wilkinson,A.J.,and Ogura: "Probing the mechanism of ATP hydrolysis and substrate translocation in the AAA protease FtsH by modelling and mutagenesis."Mol.Microbiol.. 39. 890-903 (2000)

Karata, K.、Verma, C.S.、Wilkinson, A.J. 和 Ogura：“通过建模和诱变探讨 AAA 蛋白酶 FtsH 中 ATP 水解和底物易位的机制。”Mol.Microbiol.. 39. 890-903 (2000)

小椋光,龍田高志,友安俊文: "分子シャペロンによる細胞機能制御(永田和宏,森正敬,吉田賢右(編))シュプリンガー・フェアラークフ"大腸菌のストレス応答と分子シャペロン.(印刷中). (2001)

Hikaru Ogura、Tatsuta、Toshifumi Tomoyasu：“分子伴侣控制细胞功能（Kazuhiro Nagata、Masataka Mori、Kensuke Yoshida（编辑））Springer-Verlaghoff”大肠杆菌中的应激反应和分子伴侣（正在出版）。 2001））

Tatsuta,T.,Joo,D.M.,Calendar,R.,Akiyama,Y.,and Ogura,T.: "Evidence for an active role of the DnaK chaperone system in the degradation of σ^<32>."FEBS Lett.. 478. 271-275 (2000)

Tatsuta, T.、Joo, D.M.、Calendar, R.、Akiyama, Y. 和 Ogura, T.：“DnaK 伴侣系统在 FEBS Lett 降解中发挥积极作用的证据。” 478.271-275（2000）

Tatsuta, T., Joo, D.M., Calendar, R., Akiyama, T., and Ogura, T.: "Evidence for an active role of the DnaK chaperone system in the degradation of σ^<32>."FEBS Lett.. 478. 271-275 (2000)

Tatsuta, T.、Joo, D.M.、Calendar, R.、Akiyama, T. 和 Ogura, T.：“DnaK 伴侣系统在 σ^<32> 降解中发挥积极作用的证据。”FEBS Lett。 478.271-275（2000）

小椋光,唐田清伸,千葉志信,秋山芳展: "AAAプロテアーゼ"蛋白質核酸酵素(臨時増刊号「新世紀に向けての蛋白質科学の進展」(中村春木,森川耿右,鈴木紘一,三浦謹一郎(編)). (印刷中). (2001)

Hikaru Ogura、Kiyonobu Karata、Shinobu Chiba、Yoshinobu Akiyama：“AAA 蛋白酶”蛋白质核酸酶（特刊“迈向新世纪的蛋白质科学进展”）（Haruki Nakamura、Kosuke Morikawa、Koichi Suzuki、Kinichiro Miura）（ed. ）（正在出版）。