Evolutionarily Conserved Amyloid Aggregation in the PACAP Peptide Family Is Controlled by Heparin-Sensitive Lys/Arg Gatekeeper Residues

Functional amyloid formation plays a central role in the storage of several peptide hormones, yet its structural basis remains poorly understood. Here, we investigate amyloid formation within class B1 GPCR-associated peptide hormones by analysing the aggregation propensity of the PACAP family. We show that five of six human PACAP-family peptides form amyloid fibrils not only within the pH range characteristic of secretory granules; however, aggregation is strictly dependent on the presence of glycosaminoglycans such as heparin. We identify conserved Arg/Lys-rich regions as key regulatory elements that act as conditional switches within the PACAP family. Experimental and molecular dynamics simulations show these residues function as aggregation gatekeepers but promote peptide self-assembly upon charge compensation by polyanionic cofactors. Although the glucagon and PACAP families diverged early in chordate evolution, we demonstrate, supported by seven amyloid-like crystal structures, that receptor-binding segments of PACAP peptides also function as aggregation-prone regions despite substantial sequence divergence from their counterparts in the glucagon family. Profiling aggregation in protochordate-derived peptides approximating the ancestral state of the superfamily, combined with sequence analysis of vertebrate PACAP-glucagon peptides, reveals that the intrinsic aggregation profile is remarkably conserved throughout evolution, even as receptor specificity diversifies.