Abstract
<jats:p> Post-translational modifications (PTMs) diversify neuropeptide function, yet how minimal modifications encode receptor specificity without direct contact remains a fundamental challenge in chemical biology. Pyroglutamylation (pQ), a prevalent N-terminal PTM in bioactive peptides, introduces a rigid cyclic constraint, yet its mechanistic role in receptor signaling is unclear. Here, using two newly identified endogenous <jats:italic>Aplysia</jats:italic> PRXamide receptors (ApPRXa?R1 and ApPRXa-R2) as a model system, we find that the same ligand, MMG2-pDPb (pQPPLPRYamide), produces opposite functional outcomes: pQ suppresses ApPRXa-R1 activation while enhancing ApPRXa-R2 activation. <jats:italic>In vitro</jats:italic> and <jats:italic>in silico</jats:italic> analyses demonstrate that the N-terminal pQ/Q remains solvent-exposed and does not directly contact receptor residues. Instead, pQ reshapes the ligand conformational ensemble and redistributes interaction networks across shared receptor contact sites. Strikingly, this molecular logic extends to mammalian Neuromedin U (NmU) receptors, as canine NmU (pQFLFRPRNamide) similarly biases subtype preference of human NmU receptors. Both static and dynamic analyses further reveal that receptor pocket mechanics determine the direction of this modulation: a loose and permissive pocket better accommodates the rigid pQ-constrained ligand, whereas a more compact pocket favors the non-pyroglutamylated ligand. These findings define a “PTM distal steering” mechanism that bridges “lock-key” and “induced-fit” paradigms and establish a general principle by which a minimal, non-contacting modification encodes receptor preference through ligand conformational biasing and pocket-dependent permissiveness, providing a chemical framework for optimizing stable, conformation-biased neuropeptide analogs. </jats:p>