Conformations of disulfides are conserved in inhibitory cystine knot (ICK) motif polypeptides

Abstract

The inhibitory cystine knot (ICK) motif is an evolutionarily optimized disulfide-rich peptide motif widely present in diverse phyla with distinct biological functions. Cysteine disulfides are highly conserved in the ICK motif with C1–C4 (Disulfide-I), C2–C5(Disulfide-II), and C3–C6(Disulfide-III) connectivities in a sequence. Disulfide-I and disulfide-II form a loop and the disulfide-III tethers through the loop forming a knotted fold. The current report has analysed the conformation of disulfides in the ICK motif using the side-chain torsional angles of cysteine disulfide. In crystal structures: 88% of Disulfide-I have (+,−)SynRHHook, 92% of Disulfide-II have (+,−)RHSpiral, and 100% of Disulfide-III have (−,−)LHSpiral conformations. In NMR structures, conformational diversity has been observed for each of the cysteine disulfides of the ICK motif. The highest percentage occurrence in NMR structures: 27% of Disulfide-I have (+,−)SynRHHook, 36% of Disulfide-II have (+,−)RHSpiral, and 50% of Disulfide-III have (−,−)LHSpiral conformations. In the view of the method of identification of disulfides between cysteine residues using NMR spectroscopy, the NMR structure represents an ensemble of conformations of disulfides instead of specific disulfide conformation. The retention of the conformation in both X-ray and NMR structures supports the conservation of conformation of disulfides in the ICK motif. The tendency to exhibit specific conformation of disulfide even with variations in 3D structures supports the evolutionarily optimized nature of the ICK motif.