Zhang Y L, Cramer W A
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
J Biol Chem. 1993 May 15;268(14):10176-84.
It had previously been hypothesized that the ability of a small number of immunity protein molecules in the cytoplasmic membrane to confer protection against the lethal effects of a channel-forming colicin involves a complex stabilized by electrostatic or polar interactions between immunity protein, the colicin channel, and specific sites on the cytoplasmic membrane surface defined by the presence of the tol gene translocation proteins. The hypothesis was tested (a) by constructing a hybrid colicin molecule, IaE1, containing the E1 channel domain, and the translocation and receptor domains of Ia, and (b) by altering charged residues in all peripheral regions of the immunity protein to neutral residues. It was concluded that the specificity of immunity protein requires neither specific translocation proteins, nor a specific arrangement of charged residues in the immunity protein. (c) In addition, by making 65 site-directed mutations, "immunity by-pass" mutants were found at five different loci, Ala474, Ser477, His440, Phe443, and Gly444, on two proposed membrane-spanning helices of the open colicin channel, one hydrophobic (A471-A488) and one amphiphilic (V441-W460). The mutants in the hydrophobic helix showed a larger bypass effect. The "bypass" phenotype could be assayed by (i) cytotoxicity and (ii) K+ efflux in imm+ cells caused by a bypass mutant but not wild-type colicin. It is concluded that the immunity protein exerts its specific effect through rapid lateral diffusion in the cytoplasmic membrane and helix-helix recognition and interaction with at least one hydrophobic and one amphiphilic trans-membrane helix of the colicin channel. Interaction with the amphiphilic helix implies that the immunity protein can react with the channel in the open state.
以前曾有人提出假说，即细胞质膜中少量免疫蛋白分子赋予对形成通道的大肠杆菌素致死效应的保护能力，涉及一种由免疫蛋白、大肠杆菌素通道以及由tol基因易位蛋白的存在所定义的细胞质膜表面特定位点之间的静电或极性相互作用稳定的复合物。该假说通过以下方式进行了验证：(a)构建一种杂合大肠杆菌素分子IaE1，其包含E1通道结构域以及Ia的易位和受体结构域；(b)将免疫蛋白所有外围区域的带电荷残基改变为中性残基。得出的结论是，免疫蛋白的特异性既不需要特定的易位蛋白，也不需要免疫蛋白中带电荷残基的特定排列。(c)此外，通过进行65个定点突变，在开放的大肠杆菌素通道的两条假定跨膜螺旋上的五个不同位点，即Ala474、Ser477、His440、Phe443和Gly444，发现了“免疫绕过”突变体，一条是疏水的(A471 - A488)，另一条是两亲的(V441 - W460)。疏水螺旋中的突变体显示出更大的绕过效应。“绕过”表型可通过以下方式检测：(i)细胞毒性；(ii)由绕过突变体而非野生型大肠杆菌素引起的免疫阳性细胞中的钾离子外流。得出的结论是，免疫蛋白通过在细胞质膜中的快速侧向扩散以及与大肠杆菌素通道的至少一条疏水和一条两亲跨膜螺旋的螺旋 - 螺旋识别和相互作用发挥其特异性作用。与两亲螺旋的相互作用意味着免疫蛋白可以与开放状态的通道发生反应。
