van Kraaij C, Breukink E, Noordermeer M A, Demel R A, Siezen R J, Kuipers O P, de Kruijff B
Microbial Ingredients Section, NIZO food research, Ede, The Netherlands.
Biochemistry. 1998 Nov 17;37(46):16033-40. doi: 10.1021/bi980931b.
Nisin is an amphiphilic peptide with a strong antimicrobial activity against various Gram-positive bacteria. Its activity results from permeabilization of bacterial membranes, causing efflux of cytoplasmic compounds. To get information on the molecular mechanism of membrane permeabilization, a mutant of nisin Z containing the C-terminal extension Asp-(His)6 was produced. The biological and anionic lipid-dependent membrane activity of this peptide was very similar to that of nisin Z. Analysis of the pH dependence of model membrane interactions with the elongated peptide indicated the importance of electrostatic interactions of the C-terminus with the target membrane for membrane permeabilization. Most importantly, the membrane topology of the C-terminus of the molecule could be determined by trypsin digestion experiments, in which trypsin was encapsulated in the lumen of large unilamellar vesicles. The results show that the C-terminal part of the peptide translocates across model membranes. The pH and anionic lipid dependence of translocation closely paralleled the results of membrane permeabilization studies. Binding of nickel ions to the histidines blocked translocation of the C-terminus and concomitantly resulted in a 4-fold reduced capacity to induce K+ leakage. The results demonstrate for the first time that pore formation of nisin involves translocation of the C-terminal region of the molecule across the membrane.
乳链菌肽是一种两亲性肽，对多种革兰氏阳性菌具有强大的抗菌活性。其活性源于细菌细胞膜的通透性改变，导致细胞质化合物外流。为了解膜通透性的分子机制，制备了一种含有C端延伸序列Asp-(His)6的乳链菌肽Z突变体。该肽的生物学活性和依赖阴离子脂质的膜活性与乳链菌肽Z非常相似。对模型膜与延长肽相互作用的pH依赖性分析表明，C端与靶膜的静电相互作用对膜通透性很重要。最重要的是，分子C端的膜拓扑结构可以通过胰蛋白酶消化实验来确定，在该实验中，胰蛋白酶被包裹在大单层囊泡的内腔中。结果表明，该肽的C端部分可跨模型膜转运。转运的pH和阴离子脂质依赖性与膜通透性研究结果密切平行。镍离子与组氨酸结合会阻断C端的转运，并同时导致诱导K+泄漏的能力降低4倍。结果首次证明，乳链菌肽的孔形成涉及分子C端区域跨膜转运。
