LAUSR

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.