Abstract
Cyclotides have attracted great interest as drug design scaffolds because of their unique cyclic cystine knotted topology. They are classified into three subfamilies, among which the bracelet subfamily represents the majority and comprises the most bioactive cyclotides, but are the most poorly utilized in drug design applications. A long-standing challenge has been the very low in vitro folding yields of bracelets, hampering efforts to characterize their structures and activities. Herein, we report substantial increases in bracelet folding yields enabled by a single point mutation of residue Ile-11 to Leu or Gly. We applied this discovery to synthesize mirror image enantiomers and used quasi-racemic crystallography to elucidate the first crystal structures of bracelet cyclotides. This study provides a facile strategy to produce bracelet cyclotides, leading to a general method to easily access their atomic resolution structures and providing a basis for development of biotechnological applications.
Highlights
Cyclotides are a large family of topologically unique peptides characterized by a head-to-tail cyclic backbone and three interlocked disulfide bonds [1,2]
The [I11L] mutants exhibited improved folding yields and retained the membrane binding ability compared to their wild-type counterparts
We propose these Leu mutants could be surrogates of the native peptides to be used in agricultural or therapeutic applications, in which cost-effective production is a major concern
Summary
Cyclotides are a large family of topologically unique peptides characterized by a head-to-tail cyclic backbone and three interlocked disulfide bonds [1,2]. This combination of structural features forms a cyclic cystine knot (CCK, Figure 1a) motif and confers cyclotides exceptional stability [3]. Correct folding of their structures is thought to be essential for cyclotides to carry out their natural plant defense functions against insects [4] and pathogenic fungi [5], as well as many other bioactivities suited for applications in agriculture and medicine [2].
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