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

Reviewer #1 (Public review):

Summary:

The study identifies two previously uncharacterized endogenous receptors for Aplysia PRXamide peptides and shows that N-terminal pyroglutamylation can produce opposite effects on receptor activation. The authors further propose that this modification acts indirectly by altering peptide conformation and that receptor pocket properties determine the direction of its effect. The findings are potentially significant for understanding how peptide modifications influence receptor selectivity, but the evidence supporting the proposed molecular mechanism is not yet sufficiently strong.

Strengths:

The identification and functional characterization of the two receptors are valuable. The contrasting effects of pyroglutamylation, together with peptide and receptor mutagenesis and computational analyses, provide an interesting framework for investigating peptide-receptor selectivity.

Weaknesses:

Most of the statements about novelty and generality are stronger than warranted by the current data. The central mechanistic model relies heavily on computational predictions and indirect functional measurements, without direct structural or receptor-proximal evidence. In addition, the human receptor data provide only partial support for the proposed mechanism, particularly because the pyroglutamylated and non-pyroglutamylated peptides are not significantly different at human receptor 2. Thus, the data support differential effects of pyroglutamylation but do not yet fully establish the proposed distal conformational mechanism.

Reviewer #2 (Public review):

Summary:

The manuscript by Chang et al. presents the discovery and deorphanization of two bona fide PRXamide GPCRs, and their peptide agonists, in Aplysia californica (sea hare, a type of sea slug). Focusing on MMG2-DPb, the most potent peptide agonist, the authors demonstrate that pyroglutamination on the extracellularly facing N-terminus of the peptide reduces its agonist potency towards ApPRXa-R1 and increases potency towards ApPRXa-R2. Model-guided pocket and peptide mutagenesis demonstrate multiple differential dependencies and sensitivities of ApPRXa-R1 and ApPRXa-R2, supporting distinct mechanisms of peptide binding. The authors then show that a pyroglutaminated version of the dog Neuromedin U peptide has an increased potency towards human NMUR1, relative to the free-Gln version or the non-Gln-containing human peptide, whereas the potency towards NMUR1 is slightly reduced. These examples delineate N-terminal pyroglutamination of a peptide agonist as a receptor subtype selectivity controlling mechanism even when the peptide binds the receptor C-terminus.

Strengths:

(1) Identification of a class of GPCR-signaling peptides in a poorly characterized organism (Aplysia californica, a type of a sea slug).

(2) Deorphanization of two Aplysia californica GPCRs and the establishment of an Aplysia analog of the human Neuromedin U signaling system.

(3) A rigorous structure-function study of the Aplysia receptors and peptides.

(4) Identification of pyroglutamination as a mechanism controlling subtype selectivity in Aplysia PRXamide receptor system.

(5) Very clear and compelling graphics.

(6) Overall, the biochemical and pharmacological part of the study is strong, exciting, and well-presented.

Weaknesses:

(1) My biggest problem is with the emphasis on the 'non-contacting role' of the N-terminal pGlu: "purely on positional grounds, N-terminal pQ/Q is unlikely to form direct contacts with receptor residues", "Despite the absence of direct pQ/Q-receptor interactions...", "indirect PTM distal steering", etc. The absence of direct contacts between pGlu and the receptor residues is likely an artifact of limited precision in modeling and reflects insufficiently advanced modeling tools. The authors used Swiss-Model, Robetta, and HPEPDOCK: why not go for the state-of-the-art AI modeling software like Boltz, Chai, or AlphaFold? A quick AlphaFold3 generates a model where Gln and pGlu form perfect contacts with the receptor N-term and ECL2 (in ApPRXa-R2) or N-term and ECL3 (in ApPRXa-R1). The N-termini of both receptors are quite long, form well-defined tertiary structures, and fold onto the receptor extracellular loops; in the case of ApPRXa-R1, the N-terminal domain is stabilized by an intra-domain disulfide bond C15(NT)-C133(NT) and stapled to ECL2 via another disulfide bond C16(NT)-C322(ECL2), suggesting that these interactions are real. The 3D models generated by the authors are not available for review but based on figures, I don't see these N-terminal domains modeled at all, which would obviously affect the docked position of the peptide N-terminus and its contacts with the receptor. For example, in an AlphaFold model, ApPRXa-R1 F161, E317, R340 are in direct vicinity of the peptide's Gln / pGlu and are quite distinct in ApPRXa-R2 - direct contacts with these or nearby residues may easily explain the differential preferences for free Gln vs pGlu but none of them were tested via mutagenesis.

(2) Some conclusions and interpretations in the manuscript are overstated and lack precision. For example: "...a single N-terminal lactam modification converts a minimal chemical difference into bidirectional GPCR subtype outputs" makes the reader think about something as dramatic as an inversion of efficacy, an agonist becoming an antagonist, or at least the introduction of signaling bias, whereas in reality, the authors established that pyroglutamination reduces the potency of the peptide at one receptor but increases it at the other receptor. The use of PTMs and proteolytic processing is a broadly utilized mechanism for controlling GPCR peptide selectivity. The effects of pyroglutamination on the potency of MMG2-DPb towards its two Aplysia receptors very well align with this mechanism and should be described as such. Other examples of sentences that are similarly misleading: "... the same ligand, ..., produces opposite functional outcomes: pQ suppresses ApPRXa-R1 activation while enhancing ApPRXa-R2 activation" (in reality, both receptors are activated and the functional outcomes are the same, just achieved with different potency), "A single N-terminal lactam enables bidirectional GPCR subtype tuning...", "This bidirectional, subtype-specific effect of pQ is unprecedented in neuropeptide signaling..." (not bidirectional and not as unprecedented as the authors make it sound), etc.

(3) Overall, what the authors present as a new "indirect PTM distal steering" mechanism is really not supported by the data. The described systems fit the classical "lock-key" and "induced fit" paradigms rather than challenging them. I recommend the authors revisit their modeling approaches and model-guided interpretations of the biochemical/pharmacological data. Importantly, I do not think such revision would undermine the key strengths of the paper (listed above in Strengths). Based on experimental data alone, this is a very strong and interesting study and with more rigorous modeling and adequate interpretations, it can be made exceptional.