Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase
Individual Interactions of the b Subunits within the Stator of the Escherichia coli ATP Synthase
复制标题
DOI：
10.1074/jbc.m113.465633
复制
发表时间：
2013-08-23
影响因子：
4.8
通讯作者：
Deckers-Hebestreit, Gabriele
中科院分区：
文献类型：
--
作者：
Brandt, Karsten;Maiwald, Sarah;Deckers-Hebestreit, Gabriele
FOF1 ATP合成酶是一种旋转纳米马达，它将质子在生物膜上的易位与ATP的合成/水解结合起来。在催化过程中，由大肠杆菌中两个b亚基和δ亚基组成的外周茎抵消了中心茎旋转产生的扭矩。在这里，我们通过使用单克隆抗体和通过亚基间二硫键形成的最近邻分析来表征定子内b亚基的个体相互作用。抗体结合研究表明，其中一个b亚基的c端区域主要参与亚基delta的结合，而另一个则可与抗体结合，而不影响FOF1的功能。筛选适合于b亚基与其他定子亚基（b- α, b- β， b- δ和b-a）之间二硫交联的单独取代半胱氨酸对，并相互组合以区分两个b亚基(即b（I)和b(II)）。结果表明，b二聚体位于非催化α / β裂口，b(I)靠近α亚基，而b（II）靠近β亚基。此外，b(I)可以与亚基delta和亚基a连接。形成的亚配合物包括a-b(I)- α、b(II)- β、α -b(I)-b(II)- β和a-b(I)- δ。综上所述，所获得的数据定义了两个b亚基在非催化界面上的不同位置，并暗示每个b亚基在定子内产生稳定性方面具有不同的作用。我们认为b(I)在功能上与线粒体ATP合酶中存在的单个b亚基相关。
FOF1 ATP synthases are rotary nanomotors that couple proton translocation across biological membranes to the synthesis/hydrolysis of ATP. During catalysis, the peripheral stalk, composed of two b subunits and subunit delta in Escherichia coli, counteracts the torque generated by the rotation of the central stalk. Here we characterize individual interactions of the b subunits within the stator by use of monoclonal antibodies and nearest neighbor analyses via intersubunit disulfide bond formation. Antibody binding studies revealed that the C-terminal region of one of the two b subunits is principally involved in the binding of subunit delta, whereas the other one is accessible to antibody binding without impact on the function of FOF1. Individually substituted cysteine pairs suitable for disulfide cross-linking between the b subunits and the other stator subunits (b-alpha, b-beta, b-delta, and b-a) were screened and combined with each other to discriminate between the two b subunits (i.e. b(I) and b(II)). The results show the b dimer to be located at a non-catalytic alpha/beta cleft, with b(I) close to subunit alpha, whereas b(II) is proximal to subunit beta. Furthermore, b(I) can be linked to subunit delta as well as to subunit a. Among the subcomplexes formed were a-b(I)-alpha, b(II)-beta, alpha-b(I)-b(II)-beta, and a-b(I)-delta. Taken together, the data obtained define the different positions of the two b subunits at a non-catalytic interface and imply that each b subunit has a different role in generating stability within the stator. We suggest that b(I) is functionally related to the single b subunit present in mitochondrial ATP synthase.