Figure 9.

A potential link between precursor architecture and neofunctionality. (A) Suggested modes of duplication in precursor genes. In Viola, the precursor architecture differs between lineages. Precursors of bracelet lineages appear to contain only one modular domain (i.e., a concatenated NTR and a cyclotide domain), whereas precursors of the Möbius lineage may contain one to three such domains. The repeats in the Möbius lineage probably originated from internal gene duplication events. All precursors sharing the same architecture, in both the Möbius and bracelet lineages, probably originated from external gene duplication. (B) Sequence alignment between new types of cyclotides and previously identified archetypes. In the Möbius lineage, sequences vary between repeated cyclotide domains within the same precursor and cyclotide domains of different precursors. There are high levels of sequence conservation within repeated cyclotide domains, as exemplified by kB1 and kS within Vok1. However, the sequences of cyclotide domains in precursors containing multiple cyclotide domains in general are frequently very different, as exemplified by kalata S and viul F within prc-Viul F[C]-FS4U, and viba 19 and viba 21 within VbCP14. Of the precursor sequences shown in the figure, the molecular species YS7, which belongs to the Mobius lineage, has a similar sequence to archetypical bracelet cyclotides. For example, valta3-YS4 lacks a proline residue in loop 5 and has an elongated loop 3. Conversely, the molecular species FA1 contains lipophilic residues in loops 2 and 5, while loop 3 consists mainly of negatively charged residues. (C) Comparison of cyclotides' surfaces and electrostatic potentials. New types of cyclotides— viman-FA1, viman1-RS2, and valta3-YS4, and viul-F—are shown with their electrostatic potential surfaces. The distributions of their electrostatic potentials are generally dissimilar to those seen in typical bracelet and Mobius cyclotides. Residues with high exposed ratios are indicated by solid and dashed lines on the molecular surfaces. Residues highlighted with solid lines have dissimilar physicochemical properties to those found in the corresponding positions of archetypical cyclotides. Residues whose physicochemical properties match those of the archetypical cyclotides are indicated by dotted lines. Residues are numbered in accordance with their sequence positions in (B). Negatively charged surface regions are colored in red, positively charged regions in blue, and hydrophobic regions in green. The numbering of residues follow the consensus sequence of the cyclotide domain in Figure 3.