Cyclotides are plant-derived peptides that contain three interlocking disulfide bonds, six inter-cysteine loops and a head-to-tail cyclic backbone. Their unique cyclic cystine knotted topology confers them phenomenal stability against proteolytic degradation, and hence allows their use as drug design scaffolds to stabilise peptide pharmacophores with clinical potentials. Cyclotides are classified into three subfamilies, among which the bracelet subfamily is the largest and comprises the most bioactive cyclotides, including cycloviolacin O2 and hyen D,1 but are the most poorly utilised in drug design applications. The structure-activity relationship studies of bracelet cyclotides are hampered by the fact that only low in vitro folding yields can be achieved under the standard cyclotide folding condition.
Here, we focus on understanding the in vitro folding of bracelet cycloitdes and report substantial increases enhanced by a single point mutation of the third residue in loop 2, Ile-11, to Leu or Gly. These single mutations were introduced into four examplar bracelet cyclotides, i.e., cycloviolacin O2 and O9, hyen D, and kalata B5, proving that the single substitution might have broader utility. We further applied this discovery to synthesise mirror image enantiomers and use quasi-racemic crystallography to elucidate the first crystal structures of cycloviolacin O2 and hyen D. In summary, our study provides a facile approach to produce bracelet cyclotides, leading to a general method to easily access the atomic resolution structures and providing a basis for the development of bracelet scaffolds in biotechnological applications.