van Raaij M J, Orriss G L, Montgomery M G, Runswick M J, Fearnley I M, Skehel J M, Walker J E
MRC Laboratory of Molecular Biology, Cambridge, U.K.
Biochemistry. 1996 Dec 10;35(49):15618-25. doi: 10.1021/bi960628f.
The mitochondrial ATPase inhibitor subunit is a basic protein of 84 amino acids that helps to regulate the activity of F1F0-ATPase. In order to obtain structural information on the mechanism of inhibition, the bovine inhibitor subunit has been expressed in Escherichia coli and purified in high yield. The recombinant protein has a similar inhibitory activity to the inhibitor subunit isolated from bovine mitochondria. Progressive N-terminal and C-terminal deletion mutants of the inhibitor subunit have been produced either by overexpression and purification, or by chemical synthesis. By assaying the truncated proteins for inhibitory activity, the minimal inhibitory sequence of the inhibitor subunit has been defined as consisting of residues 14-47. The immediately adjacent sequences 10-13 and 48-56 help to stabilize the complex between F1F0-ATPase and the inhibitor protein, and residues 1-9 and 57-84 appear to be dispensable. At physiological pH values, the inhibitor subunit is mainly alpha-helical and forms monodisperse aggregates in solution. Smaller inhibitory fragments of the inhibitor protein, such as residues 10-50, seem to have a mainly random coil structure in solution, but they can adopt the correct inhibitory conformation when they from a complex with the ATPase. However, these latter fragments are mainly monomeric in solution, suggesting that the aggregation of the inhibitor subunit in solution may be due to intermolecular alpha-helical coiled-coil formation via the C-terminal region. The noninhibitory peptides consisting of residues 10-40 and 23-84 of the inhibitor protein can bind to F1F0-ATPase, and interfere with inhibition by the intact inhibitor subunit. The noninhibitory fragments of the inhibitor protein consisting of residues 22-46 and 44-84 do not compete with the inhibitor subunit for its binding site on F1F0-ATPase.
线粒体ATP酶抑制亚基是一种由84个氨基酸组成的碱性蛋白质，有助于调节F1F0 - ATP酶的活性。为了获得关于抑制机制的结构信息，牛抑制亚基已在大肠杆菌中表达并以高产率纯化。重组蛋白具有与从牛线粒体分离的抑制亚基相似的抑制活性。通过过表达和纯化或化学合成产生了抑制亚基的渐进性N端和C端缺失突变体。通过测定截短蛋白的抑制活性，抑制亚基的最小抑制序列已被定义为由14 - 47位残基组成。紧邻的序列10 - 13和48 - 56有助于稳定F1F0 - ATP酶与抑制蛋白之间的复合物，而1 - 9位和57 - 84位残基似乎是可有可无的。在生理pH值下，抑制亚基主要呈α螺旋结构，并在溶液中形成单分散聚集体。抑制蛋白的较小抑制片段，如10 - 50位残基，在溶液中似乎主要具有无规卷曲结构，但当它们与ATP酶形成复合物时可以采用正确的抑制构象。然而，这些后一种片段在溶液中主要是单体，这表明抑制亚基在溶液中的聚集可能是由于通过C端区域形成分子间α螺旋卷曲螺旋。由抑制蛋白的10 - 40位和23 - 84位残基组成的非抑制性肽可以与F1F0 - ATP酶结合，并干扰完整抑制亚基的抑制作用。由22 - 46位和44 - 84位残基组成的抑制蛋白的非抑制片段不与抑制亚基竞争其在F1F0 - ATP酶上的结合位点。
