Duché D, Parker M W, González-Mañas J M, Pattus F, Baty D
Laboratoire d'Ingénierie et de Dynamique des Systèmes Membranaires, Centre de Biochimie et de Biologie Moléculaire, Marseille, France.
J Biol Chem. 1994 Mar 4;269(9):6332-9.
Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 alpha-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.
在大肠杆菌素A的成孔结构域中设计了四个二硫键，以探究与其膜插入和通道形成相关的构象变化。可溶性成孔结构域由10个α螺旋组成，有两层外层螺旋（分别为螺旋1、2和3 - 7）夹着中间一层由三个螺旋（8 - 10）组成的结构。螺旋8和9形成一个发夹结构，完全被掩埋，且仅由疏水和中性残基组成。这个螺旋发夹结构被推测为膜锚定结构。每个具有单个二硫键的双半胱氨酸突变体，分别交联螺旋1与9（H1/H9）、5与6（H5/H6）、7与8（H7/H8）或9与10（H9/H10），均无法促进敏感大肠杆菌细胞中的钾离子外流。加入还原剂可恢复活性。用溴化脂质囊泡和平面脂质双层进行的体外研究表明，连接螺旋1与9的二硫键会阻止大肠杆菌素A插入膜中，而其他二硫键突变体则很容易插入脂质囊泡。在存在膜电位的情况下，所有设计的桥连都阻止了导电通道的形成。这种新方法表明膜插入和通道形成是两个独立的步骤。此外，不同二硫键引入的距离限制对大肠杆菌素A活性的影响表明，螺旋对1和2在膜插入时会与其他螺旋分开。螺旋3 - 10仍保持在一起。因此，结果表明螺旋发夹结构与膜表面平行。相比之下，膜电位诱导大肠杆菌素通道需要螺旋关联的深刻重组。根据几种提出的膜结合大肠杆菌素和通道结构模型对这些结果进行了讨论。
