Structure and molecular interaction of ion-translocating subunits of Na+-coupled V-ATPase

Na耦合V-ATP酶离子易位亚基的结构和分子相互作用

• 批准号: 15570108
• 负责人: KAKINUMA Yoshimi
• 金额: $ 2.37万
• 依托单位: Ehime University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15570108/
• 关键词: V-ATPase; Sodium; Ion; Enterococcus hirae

(i)We found that the H626 and E634 residues in the vicinity of the membrane surface of the seventh transmembrane helix of NtpI subunit are highly conserved among those of various V-ATPases. The V-ATPases activity totally disappeared in the H626R or E634Q mutant, suggesting the functional importance of these residue in NtpI.(ii)The acidic residue E139 (kE139) which exists in the center of the fourth transmembrane helix of NtpK subunit is essential for ion binding. In purfied kE139D mutant enzyme, the affinity for Na+ of the ATP hydrolytic activity decreased about 1/4 of that of the wild-type enzyme. The kV138L mutation was isolated as the partial suppressor mutation for kE139D mutation. Site-directed mutagenesis at kV138 residue revealed that the kV138M/kE139D double mutant fully recovered the ATPase activity like that of the wild-type enzyme. These results suggest that spatial arrangement of the carboxyl group of kE139 is strictly required for Na+ binding in E.hirae V-ATPase.(iii)We found that the nucleotide binding site exists in NtpB subunit with the photoaffinity label experiment of ATP.(iv)We succeeded in purification NtpK subunit ring and found that the ion-binding rotary rotor consists of NtpK heptamer by biochemical analysis and electron microscope image analysis.

(i)We发现NtpI亚基第七跨膜螺旋膜表面附近的H626和E634残基在各种V-ATP酶中高度保守。在H626 R或E634 Q突变体中，V-ATP酶活性完全消失，表明这些残基在NtpI中的功能重要性。（ii）存在于NtpK亚基第四跨膜螺旋中心的酸性残基E139（kE 139）是离子结合所必需的。在纯化的kE 139 D突变酶中，ATP水解活性的Na+亲和力降低了野生型酶的约1/4。kV 138 L突变被分离为kE 139 D突变的部分抑制突变。在kV 138残基的定点突变显示，kV 138 M/kE 139 D双突变体完全恢复了与野生型酶一样的ATP酶活性。这些结果表明，kE 139的羧基的空间排列是严格需要的Na+结合在E. hienovirus V-ATP酶。（iii）通过ATP的光亲和标记实验，发现NtpB亚基上存在核苷酸结合位点。（iv）成功地纯化了NtpK亚基环，通过生化分析和电镜图像分析，发现NtpK亚基环上的离子结合旋转转子由NtpK七聚体组成。

项目成果

Identification of nucleotide binding sites in V-type Na+-ATPase from Enterococcus hirae

海拉肠球菌 V 型 Na -ATP 酶核苷酸结合位点的鉴定

• 发表时间: 2004
• 期刊: Bioscience, Biotechnology, and Biochemistry 68
• 作者: T.Hosaka;T.Murata;Y.Kakinuma;I.Yamato
• 通讯作者: I.Yamato

Murata, T. et al.: "The membrane domain of the Na+motive V-ATPase from Enterococcus hirae contains a heptameric rotor"Journal of Biological Chemistry. 278. 21162-21167 (2003)

Murata, T. 等人：“来自海拉肠球菌的 Na 动力 V-ATP 酶的膜结构域包含七聚转子”《生物化学杂志》。

The membrane domain of the Na+-motive V-ATPase from Enterococcus hirae contains a heptameric rotor

• DOI: 10.1074/jbc.m301620200
• 发表时间: 2003-06-06
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Murata, T;Arechaga, I;Walker, JE
• 通讯作者: Walker, JE