Bu Zimei, Wang Ligong, Kendall Debra A
Department of Molecular and Cell Biology, University of Connecticut, 91 N. Eagleville Road, Storrs, CT 06269-3125, USA.
J Mol Biol. 2003 Sep 5;332(1):23-30. doi: 10.1016/s0022-2836(03)00840-4.
In Escherichia coli, SecA is a large, multifunctional protein that is a vital component of the general protein secretion pathway. In its membrane-bound form it functions as the motor component of the protein translocase, perhaps through successive rounds of membrane insertion and ATP hydrolysis. To understand both the energy conversion process and translocase assembly, we have used contrast-matched, small-angle neutron-scattering (SANS) experiments to examine SecA in small unilamellar vesicles of E.coli phospholipids. In the absence of nucleotide, we observe a dimeric form of SecA with a radius of gyration comparable to that previously observed for SecA in solution. In contrast, the presence of either ADP or a non-hydrolyzable ATP analog induces conversion to a monomeric form. The larger radius of gyration for the ATP-bound relative to the ADP-bound form suggests the former has a more expanded global conformation. This is the first direct structural determination of SecA in a lipid bilayer. The SANS data indicate that nucleotide turnover can function as a switch of conformation of SecA in the membrane in a manner consistent with its proposed role in successive cycles of deep membrane penetration and release with concommitant preprotein insertion.
在大肠杆菌中，SecA是一种大型多功能蛋白，是一般蛋白质分泌途径的重要组成部分。以其膜结合形式，它作为蛋白质转位酶的动力组件发挥作用，可能是通过连续几轮的膜插入和ATP水解。为了理解能量转换过程和转位酶组装，我们使用对比匹配的小角中子散射（SANS）实验来研究大肠杆菌磷脂小单层囊泡中的SecA。在没有核苷酸的情况下，我们观察到SecA的二聚体形式，其回转半径与之前在溶液中观察到的SecA相当。相比之下，ADP或不可水解的ATP类似物的存在会诱导其转变为单体形式。与ADP结合形式相比，ATP结合形式的回转半径更大，这表明前者具有更扩展的整体构象。这是首次在脂质双层中对SecA进行直接结构测定。SANS数据表明，核苷酸周转可以作为SecA在膜中构象的开关，其方式与其在深度膜穿透和释放的连续循环中伴随前体蛋白插入的拟议作用一致。
