Minamino Tohru, Kazetani Ken-ichi, Tahara Aiko, Suzuki Hirofumi, Furukawa Yukio, Kihara May, Namba Keiichi
Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2006 Jul 7;360(2):510-9. doi: 10.1016/j.jmb.2006.05.010. Epub 2006 May 19.
Salmonella FliI is the flagellar ATPase which converts the energy of ATP hydrolysis into the export of flagellar proteins. It forms a ring-shaped oligomer in the presence of ATP, its analogs, or phospholipids. The extreme N-terminal region of FliI has an unstable conformation and is responsible for the interaction with other components of the export apparatus and for regulation of the catalytic mechanism. To understand the role of this N-terminal region in more detail, we used multi-angle light-scattering, analytical ultracentrifugation, far-UV CD and biochemical methods to characterize a partially functional variant of FliI, missing its first seven amino acid residues (His-FliI(Delta1-7)), whose ATPase activity is about ten times lower than that of wild-type FliI. His-FliI(Delta1-7) is monomeric in solution. The deletion increased the content of alpha-helix, suggesting that the deletion stabilizes the unstable N-terminal region into an alpha-helical conformation. The deletion did not influence the K(m) value for ATP. However, unlike the wild-type, ATP and acidic phospholipids did not induce oligomerization of His-FliI(Delta1-7) or increase its ATPase activity. These results suggest that the deletion suppresses the oligomerization of FliI, and that a conformational change in the unstable N-terminal region is required for FliI oligomerization to effectively couple the energy of ATP hydrolysis to the translocation of flagellar proteins.
沙门氏菌FliI是一种鞭毛ATP酶，它将ATP水解产生的能量转化为鞭毛蛋白的输出。在ATP、其类似物或磷脂存在的情况下，它会形成环状寡聚体。FliI的极端N端区域具有不稳定的构象，负责与输出装置的其他组分相互作用以及催化机制的调节。为了更详细地了解该N端区域的作用，我们使用多角度光散射、分析超速离心、远紫外圆二色光谱和生化方法来表征FliI的一个部分功能变体，该变体缺失其前七个氨基酸残基（His-FliI(Δ1-7)），其ATP酶活性比野生型FliI低约十倍。His-FliI(Δ1-7)在溶液中呈单体形式。缺失增加了α-螺旋的含量，表明该缺失将不稳定的N端区域稳定成α-螺旋构象。该缺失不影响ATP的米氏常数（Km）。然而，与野生型不同，ATP和酸性磷脂不会诱导His-FliI(Δ1-7)的寡聚化或增加其ATP酶活性。这些结果表明，该缺失抑制了FliI的寡聚化，并且FliI寡聚化需要不稳定的N端区域发生构象变化，以便有效地将ATP水解的能量与鞭毛蛋白的转运偶联起来。
