Cysimiditides: RiPPs with a Zn-Tetracysteine Motif and Aspartimidylation.

Aspartimidylation is a post-translational modification found in multiple families of ribosomally synthesized and post-translationally modified peptides (RiPPs). We recently reported on the imiditides, a new RiPP family in which aspartimidylation is the class-defining modification. Imiditide biosynthetic gene clusters encode a precursor protein and a methyltransferase that methylates a specific Asp residue, converting it to aspartimide. A subset of imiditides harbor a tetracysteine motif, so we have named these molecules cysimiditides. Here, using genome mining, we show that there are 56 putative cysimiditides predicted in publicly available genome sequences, all within actinomycetota. We successfully heterologously expressed two examples of cysimiditides and showed that the major products are aspartimidylated and that the tetracysteine motif is necessary for protein stability. Cysimiditides bind a Zn²⁺ ion, presumably at the tetracysteine motif. Using in vitro reconstitution of the aspartimidylation reaction, we show that Zn²⁺ is required for the methylation and subsequent aspartimidylation of the precursor protein. An AlphaFold 3 model of the cysimiditide from Thermobifida cellulosilytica shows a hairpin structure anchored by the Zn²⁺-tetracysteine motif with the aspartimide site in the hairpin loop. An AlphaFold 3 model of this cysimiditide in complex with its cognate methyltransferase suggests that the methyltransferase recognizes the Zn²⁺-tetracysteine motif to correctly dock the precursor protein. Cysimiditides expand the set of experimentally confirmed RiPPs harboring aspartimides and represent the first RiPP class that has an obligate metal ion.