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

(1)我们发现在各种V-ATPase中，NtpI亚单位第七个跨膜螺旋的膜表面附近的H626和E634残基在各种V-ATPase中高度保守。在H626R或E634Q突变体中，V-ATPase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(KE139)是离子结合所必需的。在纯化的kE139D突变酶中，ATP水解酶对Na+的亲和力约为野生型酶的1/4。分离到kV138L突变作为kE139D突变的部分抑制突变。对kV138残基的定点突变表明，kV138M/kE139D双突变株的ATPase活性完全恢复到野生型的水平。这些结果表明KE139的羧基的空间排列严格要求Na+结合在E.hirae V-ATPase中。(Iii)通过ATP的光亲和标记实验，我们发现核苷酸结合部位存在于NtpB亚基中。(Iv)我们成功地纯化了NtpK亚单位环，并通过生化分析和电子显微镜图像分析发现，离子结合的旋转转子由NtpK七聚体组成。

(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.