Cyclotide Structures Revealed by NMR, with a Little Help from X‐ray Crystallography

This review highlights the predominant role that NMR has had in determining the structures of cyclotides, a fascinating class of macrocyclic peptides found in plants. Cyclotides contain a cystine knot, a compact structural motif that is constrained by three disulfide bonds and able to resist chemical and biological degradation. Their resistance to proteolytic degradation has made cyclotides appealing as drug leads. Herein, we examine the developments that led to the identification and conclusive determination of the disulfide connectivity of cyclotides and describe in detail the structural features of exemplar cyclotides. We also review the role that X‐ray crystallography has played in resolving cyclotide structures and describe how racemic crystallography opened up the possibility of obtaining previously inaccessible X‐ray structures of cyclotides. Untying the knot: We explore the role that NMR spectroscopy has played in the solution of cyclotide structures, with a focus on the disulfide connectivity of the cystine knot. The cystine knot is a tight arrangement of three disulfide bonds at the core of cyclotides which contributes to their exceptional stability and to their appeal as therapeutic agents.