Tertiary structure of a Nif11-like leader peptide mediates protein-protein interactions with a RiPP biosynthetic...

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced from precursor peptides that typically contain a short, unstructured N-terminal leader region that engages RiPP biosynthetic enzymes through peptide-protein contacts. A distinct subclass of leader peptides, the nitrogen-fixing protein 11-like leader peptides (Nif11-LPs), are unusually long and conspicuously enriched in cyanobacteria. No atomic structures of Nif11-LPs have been reported. Here, we report the solution structure of MppEleader, a Nif11-LP from the marine cyanobacterium Moorena producens PAL. MppEleader adopts a bipartite architecture with a rigid N-terminal tetrahelical bundle stabilized around a hydrophobic nucleus, followed by a dynamic, largely disordered C-terminal region containing a conserved L(X)₄L motif. We show that the α-ketoglutarate/non-heme iron enzyme MppG dihydroxylates the macrocyclized MppEcore peptide in a strictly leader-dependent manner. NMR titrations and structure-guided mutagenesis demonstrate that both the tetrahelical hydrophobic nucleus and the L(X)₄L motif of MppE make direct, functionally required contacts with MppG, including its RiPP recognition element (RRE) domain. An AlphaFold3-guided structural model rationalizes these contacts. Together, these findings establish a tertiary structure-dependent protein-protein interaction paradigm for RiPP core peptide modification, distinguishing Nif11-LP recognition from canonical short-leader mechanisms and expanding the understanding of substrate recognition in RiPP biosynthesis.