Shape Shifting Proteusin Peptides and Their Multimodal Binding Interactions Underlie RiPP Biosynthesis

Abstract Ribosomally synthesized and post-translationally modified peptides (RiPPs) are produced through the selective recognition and enzymatic modification of precursor peptides. While canonical RiPP leader peptides are generally short and unstructured, the discovery of nitrile hydratase-like leader peptides (NHLPs) has revealed an unexpected class of large, protein-like leaders with a tertiary fold that expand the structural and mechanistic diversity of RiPP biosynthesis. Here, we review recent structural and biochemical studies of NHLPs and their interactions with diverse RiPP biosynthetic enzymes. These studies reveal that NHLPs engage their cognate enzymes through distinct interaction modes, involving different combinations of structured and disordered regions of the leader. Together, these findings establish NHLPs as a unique paradigm for substrate recognition that bridges conventional peptide−protein and protein−protein interactions in natural product biosynthesis. We discuss the emerging principles governing NHLP−enzyme recognition, outstanding questions regarding these interactions, and their implications for the discovery and engineering of RiPP biosynthetic pathways.