Abdul Ghani Hafiza, Henriques Sónia Troeira, Huang Yen-Hua, Swedberg Joakim E, Schroeder Christina I, Craik David J
Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072, Australia.
Biopolymers. 2017 Jan;108(1). doi: 10.1002/bip.22927.
Cyclotides are plant-derived host defense peptides displaying exceptional stability due to their cyclic cystine knot comprising three intertwined disulfide bonds and a cyclic backbone. Their six conserved cysteine residues are separated by backbone loops with diverse sequences. Prototypical cyclotides from the Möbius (kalata B1) and trypsin inhibitor (MCoTI-II) subfamilies lack sequence homology with one another, but both are able to penetrate cells, apparently via different mechanisms. To delineate the influence of the sequences of the loops on the structure and cell internalization of these two cyclotide subfamilies, a series of Möbius/trypsin inhibitor loop-chimeras of kalata B1 and MCoTI-II were synthesized, and structurally and functionally characterized. NMR analysis showed that the structural fold of the majority of chimeric peptides was minimally affected by the loop substitutions. Substituting loops 3, 5, or 6 of MCoTI-II into the corresponding loops of kalata B1 attenuated its hemolytic and cytotoxic activities, and greatly reduced its cell-penetrating properties. On the other hand, replacing loops of MCoTI-II with the corresponding loops of kalata B1 did not introduce cytotoxicity into the chimeras. Loops 2, 3, and 4 of MCoTI-II were found to contribute little to cell-penetrating properties. Overall, this study provides valuable insights into the structural basis for the hemolytic, cytotoxic, and cell-penetrating properties of kalata B1 and MCoTI-II, which could be useful for future engineering of cyclotides to carry bioactive epitopes to intracellular targets.
环肽是植物源宿主防御肽，因其由三个相互缠绕的二硫键和一个环状主链组成的环胱氨酸结而具有非凡的稳定性。它们的六个保守半胱氨酸残基被具有不同序列的主链环隔开。来自莫比乌斯环（卡拉塔B1）和胰蛋白酶抑制剂（MCoTI-II）亚家族的典型环肽彼此缺乏序列同源性，但两者都能够穿透细胞，显然是通过不同的机制。为了阐明环对这两个环肽亚家族的结构和细胞内化的影响，合成了一系列卡拉塔B1和MCoTI-II的莫比乌斯环/胰蛋白酶抑制剂环嵌合体，并对其进行了结构和功能表征。核磁共振分析表明，大多数嵌合肽的结构折叠受环替换的影响最小。将MCoTI-II的环3、环5或环6替换为卡拉塔B1的相应环会减弱其溶血和细胞毒性活性，并大大降低其细胞穿透特性。另一方面，用卡拉塔B1的相应环替换MCoTI-II的环不会给嵌合体引入细胞毒性。发现MCoTI-II的环2、环3和环4对细胞穿透特性贡献不大。总体而言，这项研究为卡拉塔B1和MCoTI-II的溶血、细胞毒性和细胞穿透特性的结构基础提供了有价值的见解，这可能有助于未来环肽工程将生物活性表位携带到细胞内靶点。
