Decoding of amidated aromatic C-terminus and sulfation by cholecystokinin receptors reveals conserved and divergent...

Understanding how evolutionarily-related receptors preserve recognition principles for conserved peptide post-translational modifications (PTMs) while acquiring new selectivity remains central to neuropeptide-G protein-coupled receptor (GPCR) biology. The Aplysia cholecystokinin (CCK) system provides an informative model, as its peptides combine two representative PTM-related features: an amidated aromatic C-terminus (RFamide/DFamide), and a dual-tyrosine sulfation pattern exceeding the single-site architecture typical of vertebrates. Notably, its two receptor subtypes, apCCKR1 and apCCKR2, exhibit distinct sensitivities to ligands carrying different numbers of sulfated tyrosines, yet the molecular basis remains unclear. Here, we first used multiple docking approaches and found that CCK ligands could insert into receptor binding pockets in either N-terminal-in or C-terminal-in orientations. We then applied pose-specific mutagenesis to resolve this ambiguity and established a common C-terminal-in binding mode for apCCK peptides, similar to mammalian CCK/gastrin binding modes observed by cryo-EM. Within this framework, recognition of the C-terminal aromatic motif is hierarchically organized through three receptor positions, comprising a conserved anchor, a CCK-biased layer and a flexible tuning site. Moreover, we uncovered a mechanism for sulfation-number-dependent subtype selectivity: apCCKR1 accommodates an additional sulfate with minimal structural rearrangement, whereas apCCKR2 loses a favorable electrostatic network, disfavoring the disulfated ligand. Comparative analyses across representative lineages identified a conserved sulfate-recognition module alongside a lineage-restricted modifier, enabling inferences regarding the ancestral state of sulfation recognition and how it was differentially remodeled across protostomes and deuterostomes. Together, these findings establish a framework for understanding how homologous receptors may decode peptide chemical features through conserved structural logic and adaptive diversification.