Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains.

Nonribosomal peptide synthetases (NRPSs) catalyze the formation of structurally diverse and biologically important peptides. Given their modular organization, NRPSs provide an enormous potential for biocombinatorial approaches to generate novel bioactive compounds. Crucial for the exploitation of this potential is a profound knowledge of the intermolecular communication between partner NRPSs. The overall goal of this study was to understand the basis of protein-protein communication that facilitates the selective interaction in these multienzyme complexes. On this account, we studied the relevance of short regions at the termini of the NRPSs tyrocidine (Tyc) synthetases TycA, TycB, and TycC, constituting the Tyc biosynthetic template. In vitro and in vivo investigations of C-terminal deletion mutants of the initiation module TycA provided evidence for the existence and impact of short communication-mediating (COM) domains. Their decisive role in protein-protein recognition was subsequently proven by means of COM domain-swapping experiments. Substitution of the terminal COM domains between the donor modules TycA and TycB3, as well as between the acceptor modules TycB1 and TycC1, clearly demonstrated that matching pairs of COM domains are both necessary and sufficient for the establishment of communication between partner NRPSs in trans. These results corroborated the generality of COM domains, which were subsequently exploited to induce crosstalk, even between NRPSs derived from different biosynthetic systems. In conclusion, COM domains represent interesting tools for biocombinatorial approaches, which, for example, could be used for the generation of innovative natural product derivatives.