Cooperation between a T Domain and a Minimal C‐Terminal Docking Domain to Enable Specific Assembly in a Multiprotein NRPS

Non‐ribosomal peptide synthetases (NRPS) produce natural products from amino acid building blocks. They often consist of multiple polypeptide chains which assemble in a specific linear order via specialized N‐ and C‐terminal docking domains (N/CDDs). Typically, docking domains function independently from other domains in NRPS assembly. Thus, docking domain replacements enable the assembly of “designer” NRPS from proteins that normally do not interact. The multiprotein “peptide‐antimicrobial‐Xenorhabdus” (PAX) peptide‐producing PaxS NRPS is assembled from the three proteins PaxA, PaxB and PaxC. Herein, we show that the small CDD of PaxA cooperates with its preceding thiolation (T1) domain to bind the NDD of PaxB with very high affinity, establishing a structural and thermodynamical basis for this unprecedented docking interaction, and we test its functional importance in vivo in a truncated PaxS assembly line. Similar docking interactions are apparently present in other NRPS systems. The interaction between two non‐ribosomal peptide synthetase (NRPS) proteins mediates the biosynthesis of PAX peptides and involves an extended docking interface, where a part of the thiolation (T) domain and a minimal C‐terminal docking domain (CDD) cooperate to bind to the N‐terminal docking domain (NDD) with nanomolar affinity—the highest docking domain affinity yet observed in such megasynthases.