Dempsey Brian R, Economou Anastassios, Dunn Stanley D, Shilton Brian H
Department of Biochemistry, University of Western Ontario, London, ON N6A 5C1, Canada.
J Mol Biol. 2002 Jan 25;315(4):831-43. doi: 10.1006/jmbi.2001.5279.
Preprotein translocase is a general and essential system for bacterial protein export, the minimal components of which are SecA and SecYEG. SecA is a peripheral ATPase that associates with nucleotide, preprotein, and the membrane integral SecYEG to form a translocation-competent complex. SecA can be separated into two domains: an N-terminal 68 kDa ATPase domain (N68) that binds preprotein and catalyzes ATP hydrolysis, and a 34 kDa C-terminal domain that regulates the ATPase activity of N68 and mediates dimerization. We have carried out gel filtration chromatography, analytical ultracentrifugation, and small-angle X-ray scattering (SAXS) to demonstrate that isolated N68 self-associates to form a tetramer in solution, indicating that removal of the C-terminal domain facilitates the formation of a higher-order SecA structure. The associative process is best modelled as a monomer-tetramer equilibrium, with a K(D) value of 63 microM(3) (where K(D)=monomer/[tetramer]) so that at moderate concentrations (10 microM and above), the tetramer is the major species in solution. Hydrodynamic properties of the N68 monomer indicate that it is almost globular in shape, but the N68 tetramer has a more ellipsoidal structure. Analysis of SAXS data indicates that the N68 tetramer is a flattened, bi-lobed structure with dimensions of approximately 13.5 nm x 9.0 nm x 6.5 nm, that appears to contain a central pore.
前体蛋白转位酶是细菌蛋白质输出的一个普遍且必需的系统，其最小组成部分是SecA和SecYEG。SecA是一种外周ATP酶，它与核苷酸、前体蛋白以及膜整合蛋白SecYEG结合，形成一个具有转位能力的复合物。SecA可分为两个结构域：一个N端68 kDa的ATP酶结构域（N68），它结合前体蛋白并催化ATP水解；以及一个34 kDa的C端结构域，它调节N68的ATP酶活性并介导二聚化。我们进行了凝胶过滤色谱、分析超速离心和小角X射线散射（SAXS）实验，以证明分离出的N68在溶液中自缔合形成四聚体，这表明去除C端结构域有助于形成更高阶的SecA结构。缔合过程最好被模拟为单体 - 四聚体平衡，解离常数K(D)值为63 μM³（其中K(D)=[单体]⁴/[四聚体]），因此在中等浓度（10 μM及以上）时，四聚体是溶液中的主要形式。N68单体的流体动力学性质表明它几乎呈球形，但N68四聚体具有更椭圆的结构。对SAXS数据的分析表明，N68四聚体是一种扁平的双叶结构，尺寸约为13.5 nm×9.0 nm×6.5 nm，似乎含有一个中央孔。
