Distal conformational steering by N-terminal pyroglutamylation enables subtype-selective GPCR activation across Aplysia PRXamide and human Neuromedin U signaling

Technical Summary: Distal Conformational Steering by N-terminal Pyroglutamylation in GPCR Signaling

Problem Statement
Post-translational modifications (PTMs) are known to diversify neuropeptide function and regulate G protein-coupled receptor (GPCR) signaling. While canonical models suggest PTMs modulate activity through direct ligand-receptor contacts, the mechanistic basis for how specific modifications, such as N-terminal pyroglutamylation (pQ), can exert subtype-selective effects without directly engaging receptor residues remains unclear. Specifically, it is unknown how a minimal, non-contacting chemical modification can encode receptor preference or produce opposing functional outcomes across different receptor subtypes.

Methodology
The study employed an integrated approach combining endogenous neuropeptide discovery, functional pharmacology, structural modeling, and molecular dynamics:

1. System Establishment: Liquid chromatography-mass spectrometry (LC-MS/MS) confirmed the endogenous coexistence of pyroglutamylated (MMG2-pDPb) and non-pyroglutamylated (MMG2-DPb) PRXamide peptides in Aplysia californica. Bioinformatics and phylogenetic analyses identified two novel Aplysia PRXamide receptors (ApPRXa-R1 and ApPRXa-R2).
2. Functional Pharmacology: Concentration-response curves were generated using IP1 accumulation assays in CHO-K1 cells to quantify ligand potency (EC50) and efficacy (Emax) for both peptide variants at the two receptor subtypes.
3. Structural Modeling and Docking: AI-assisted tools (HPEPDOCK, DiffDock) and homology modeling were used to generate receptor structures and dock ligand complexes. Interactions were analyzed to map contact networks.
4. Site-Directed Mutagenesis: Receptor residues predicted to interact with the ligands were mutated (e.g., to Alanine or Glutamine) to experimentally validate docking predictions and dissect the contribution of specific interaction networks to subtype selectivity.
5. Peptide Analogs: A series of MMG2-pDPb analogs were synthesized, including N-terminal substitutions (Ala, Ac-Ala), C-terminal deamidation, and alanine-scanning of residues 2–7, to define ligand-side determinants of pQ-dependent effects.
6. Cross-Species Validation: The findings were extended to mammalian systems by testing canine pQ-NmU-7 against human Neuromedin U receptors (hNmU-R1 and hNmU-R2).
7. Biophysical Analysis: Static pocket volume analysis (CASTp) and 300-ns all-atom molecular dynamics (MD) simulations were performed on human NmU receptor complexes to characterize pocket permissiveness and dynamic flexibility.

Key Results

• Bidirectional Subtype Tuning: The same N-terminal pQ modification produced opposite functional outcomes. At ApPRXa-R2, pQ enhanced ligand potency (EC50 decreased from 260 nM to 64 nM). Conversely, at ApPRXa-R1, pQ reduced potency (EC50 increased from 0.21 nM to 6.8 nM).
• Non-Contacting Mechanism: Structural modeling and mutagenesis revealed that the N-terminal pQ (and the unmodified Q) remains solvent-exposed and does not form direct contacts with receptor residues. Instead, the rigid pQ lactam ring constrains the ligand's conformational ensemble, altering the geometry and strength of interactions at shared, distal contact sites (e.g., residues in the C-terminal pharmacophore).
• Pocket Permissiveness Determines Outcome:
  • ApPRXa-R2 / hNmU-R1: These receptors possess a "loose" and permissive binding pocket. The rigid, pQ-constrained ligand is better accommodated here, strengthening key hydrophobic and polar interactions (e.g., with Y186ECL2, F210ECL2 in ApPRXa-R2), leading to enhanced activation.
  • ApPRXa-R1 / hNmU-R2: These receptors possess a "compact" and tightly optimized pocket. The rigid pQ-constrained ligand causes a conformational mismatch, disrupting the interaction network formed by the more flexible non-pQ ligand (e.g., disrupting hydrophobic packing with W432ECL3 in ApPRXa-R1), leading to reduced activation.
• Conservation: This "distal steering" logic was conserved in mammalian systems. Canine pQ-NmU-7 potentiated hNmU-R1 activation (approx. 10-fold decrease in EC50) but attenuated hNmU-R2 activation, mirroring the bidirectional regulation observed in Aplysia.
• Dynamic Evidence: MD simulations confirmed that hNmU-R1 exhibits higher radius of gyration (Rg) and residue fluctuations (RMSF) compared to the more rigid hNmU-R2, supporting the hypothesis that pocket flexibility dictates the acceptance of the constrained ligand.

Significance and Claims
The paper defines a novel "PTM distal steering" mechanism in neuropeptide-GPCR signaling. The authors claim this mechanism bridges the gap between the static "lock-key" and dynamic "induced-fit" paradigms. Specifically:

1. Mechanistic Insight: A minimal, non-contacting N-terminal modification (pQ) can encode receptor subtype preference by reshaping the ligand conformational landscape. The functional outcome (enhancement vs. suppression) is dictated not by the modification itself, but by the intrinsic "permissiveness" of the receptor's orthosteric pocket.
2. General Principle: This work establishes that receptor pocket mechanics (loose vs. compact) determine whether a conformationally constrained ligand is favored or penalized. This explains how bidirectional regulation occurs in closely related receptor subtypes.
3. Translational Potential: The findings provide a rational design framework for optimizing stable, conformation-biased neuropeptide analogs. By leveraging pQ, researchers can potentially tune pharmacokinetic stability (via resistance to aminopeptidases) and pharmacodynamic selectivity simultaneously, offering a strategy to develop subtype-selective therapeutics for targets like Neuromedin U receptors (implicated in obesity, glucose regulation, and bladder dysfunction) without requiring direct contact modifications.

The authors maintain that while pQ is known for stabilizing peptides, its role as a conformational switch for subtype-selective signaling via distal steering is a previously uncharacterized molecular logic that may be broadly applicable to other PTMs (e.g., cyclization, glycosylation) in neuropeptide systems.