Conserved cis and trans communication domains mediate module interaction in glycopeptide antibiotic NRPS assembly lines

Non-ribosomal peptide synthetases (NRPSs) assemble structurally complex natural products including clinically important antibiotics, but the mechanisms coordinating communication between multiple NRPS enzymes are incompletely understood. This work dissected the NRPS system responsible for biosynthesis of the glycopeptide antibiotic balhimycin in Amycolatopsis balhimycina to elucidate these principles.

Genetic perturbation of short terminal structural elements markedly reduced balhimycin production, demonstrating their critical biosynthetic role. AlphaFold3 predictions identified these elements as trans-COM domains that mediate specific NRPS interactions, primarily through hydrophobic contacts. Quantitative binding studies using microscale thermophoresis confirmed the importance of the trans-COM domains for multiprotein interaction, extending current models of NRPS communication.

Comparative structural analysis also uncovered a conserved class of cis-COM domains within condensation domains across GPA NRPS assembly lines. The findings establish a unified model in which trans- and cis-interfaces cooperatively maintain assembly line fidelity, redefining NRPS architecture and enabling rational engineering strategies for glycopeptide antibiotics.