Structure and dynamics of the pyroglutamylated RF-amide peptide QRFP receptor GPR103

Pyroglutamylated RF-amide peptide (QRFP) is a peptide hormone with a C-terminal RF-amide motif. QRFP selectively activates a class A G-protein-coupled receptor (GPCR) GPR103 to exert various physiological functions such as energy metabolism and appetite regulation. Here, we report the cryo-electron microscopy structure of the QRFP26-GPR103-Gq complex at 3.19 Å resolution. QRFP26 adopts an extended structure bearing no secondary structure, with its N-terminal and C-terminal sides recognized by extracellular and transmembrane domains of GPR103 respectively. This movement, reminiscent of class B1 GPCRs except for orientation and structure of the ligand, is critical for the high-affinity binding and receptor specificity of QRFP26. Mutagenesis experiments validate the functional importance of the binding mode of QRFP26 by GPR103. Structural comparisons with closely related receptors, including RY-amide peptide-recognizing GPCRs, revealed conserved and diversified peptide recognition mechanisms, providing profound insights into the biological significance of RF-amide peptides. Collectively, this study not only advances our understanding of GPCR-ligand interactions, but also paves the way for the development of novel therapeutics targeting metabolic and appetite disorders and emergency medical care.


Other minor issues:
4. There is a discrepancy between the ligand-interacting residues shown in the main Figure 3 and those shown in the Supplementary Figure 3.The authors need to determine the accurate interacting residues and show them in the figures.
Reviewer #2 (Remarks to the Author): In this study, Iwama et al. elucidated the structure of the QRFPR in complex with its ligand 26RFa and Gαq using cryo-EM.In the introduction, the authors present the family of RF-amide peptides and describe the peptides 43RFa and 26RFa and the corresponding receptor GPR103/QRFPR in more detail.In line 73, it is written, that the QRFPR is a Gαq-coupled receptor, however Ma et al. describe that GPR103 dually couples to Gαq andGαi/o proteins (doi: 10.1016/j.bbamcr.2021.119046).Can the authors comment on this?The authors describe the physiological function of GPR103 and give an overview on related peptides, such as cholecystokinin, orexin and RY-amide neuropeptide Y and knowledge on the interaction with their corresponding GPCR.In line 71/72, the authors mention that QRFP was identified by bioinformatics approach and reverse pharmacology.However, in Ref. 5, the authors described that it was identification by RT-PCR and in situ hybridization.Please, clarify and revise!In line 84, Iwama et al. introduced a GPR103 antagonist.Here, some additional information on the antagonist have to be added.
In the results section, the authors first describe the overall structure and the strategy used in this study.A C-terminally truncated variant of GPR103 was used (line 107).Please, provide data on the activity of this variant.Can the authors also comment why they used an engineered mini-Gsqi protein instead of native Gq protein?In line 126-127 the authors write: "Two disulfide bonds are observed in the GPR103 structure (Fig. 1e)" These interactions are not visible in the structure of Figure 1e.Also, these findings have to be discussed more carefully or in context with functional data, as the structure only gives information on spatial vicinity but not on physiologically relevant interactions.Further, it is described that 26RFa "adopts an elongated conformation consistent with the NMR analysis of QRFP alone" (line133-134).In this NMR study, Thuau et al. observed a nascent helix between residues 6-15 of 26RFa.Is this really consistent with the findings from the cryo-EM structure?The authors then describe the GPCR-Gα interface and write that residues within TM6 and TM7 are involved in an electrostatic network with the C-terminus of α5h (line 143/144).Here, a more detailed description have to be added.Additionally, it is of utmost importance to confirm key interaction points with mutagenesis data to proof the structural data.Contacts relevant for the function have to be distinguished from solely nearby residues.In line 146-148 it is stated, that "the bulky hydrophobic residue F151ICL2 is captured within a hydrophobic pocket in the Gα subunit, as in many other GPCRs16,21".However, in reference 21, an G protein mimetic antibody is used as substitute for the G protein.Maybe the authors can give more examples/references whether this hydrophobic interaction is known from many GPCRs and for which (including reference).The authors further describe the peptide binding site within the TM domain.Here they mention previous studies with mutant peptides (167-168) without giving any reference, this has to be added.In Figure 3b/c, it is hard to identify the relevant interactions between the C terminus of the peptide and the receptor TM core, described in lines 162-176.The authors must improve the visualization of the most important interactions or make two subfigures out of it.The authors found a unique architecture of the N-terminal region.The sentence "This N-terminal HLH motif is conserved among GPR103 homologs (Supplementary Fig. 4c), and our homology searches have failed to identify any similar sequences in other proteins, indicating unique feature of GPR103."(211-214) is a bit contradictory.Is the HLH motif unique to GPR103 or to RF-amide receptors?In line 220-236, the authors feature the N-terminal ECD configuration.Here, more detailed information is necessary.In the last part of the results section, the structure is compared to related GPCRs in "complex with Gq12-14" (line 257).However, the structure of the Y1R was determined with Gi1 protein.Please also add the corresponding PDB ID in the legend of Figure 5.It is described that "in Y1R, the nitrogen and C932.57are uniquely positioned to form a hydrogen bond (Fig. 5c), a feature not found in GPR103".This statement must be confirmed by mutagenesis studies.In line 273-278, the structures of NPY receptors are described.Here, reference 34 only includes the structure of Y1R and the respective reference describing the structure of Y1 and Y2 (Tang T et al., 2022) has to be added here.
In the discussion, the authors further analyze their findings.A more detailed analysis of the interaction between N-terminal HLH structure of GPR103 and N-terminal side of QRFP would be worth to gain new insights into this unusual interaction site.Also, it would be interesting if a Nterminal fragment of the peptide alone would be able to bind to the receptor without activating it, do the authors have any data on this?The authors describe "significant conformational changes" (line 326) within the ligand binding pocket, which are described between 1.4-1.6Å.Is the resolution of the structure high enough to predict these changes?Thus, all relevant contacts that have been found have to be confirmed by mutagenesis studies.
Receptor mutants have to be cloned replacing the respective residue by e. g. alanine, receptor expression and activation of this receptor variant has to be compared to wild type receptor.This is state-of-the art to prove relevant contacts and distinguish functionally relevant from irrelevant ones.Structural information without functional data are meaningless.Furthermore, several references are missing and wording has to be more specific in some parts.The revised parts in the text are highlighted in yellow.The major changes are as follows: • We re-analyzed the cryo-EM data using cryoSPARC and improved the resolution of the extracellular domain.As a result, we corrected our model, in which the residues 5 to 13 of QRFP26 adopt an α-helix (Fig. 1e).
• We performed thorough mutant experiments (Supplementary Fig. 1a-d and Supplementary Table 1).The expression levels of the mutants were accurately quantified by fluorescence-activated cell sorting (FACS).As most of the mutants largely reduced their expression levels, we compared their activities with that of the wild type, whose expression levels were reduced to the same extent by decreasing the amount of used plasmid in transfection.This comparison allowed us to compare and discuss the mutational effects more accurately.
• We described a more detailed structural description of the ECD containing N-terminal HLH, which was validated by mutant experiments.Thus, we have made significant changes in the description of the relevant parts.Moreover, we compared the ECD with other receptors in Fig. 7 and discussed.
• Although G-protein interactions are one of the concepts of this paper, the main topic is the mechanism of peptide ligand recognition.Moreover, as reviewer 2 pointed out, we used the highly engineered G-protein for structural analysis, while it is commonly used in the structural analysis of GPCR-G q complexes.Therefore, we have toned down the G-protein interactions, minimized references for it in the text, and depicted the interaction in Supplementary Fig. 4. We preformed thorough mutant analysis, more focusing on the ligand binding site.
• To address the requirements of reviewer 2, we performed several additional experiments, including purification of the N-terminal expression of QRFP26 and evaluation of G i /G o activity.
• Finally, we correctly cited several essential papers that were missed in the first draft.

Reviewers' comments:
Reviewer #1 (Remarks to the Author): This manuscript by Iwama etc. presents the cryo-EM structure of the RF-amide peptide receptor GPR103 in complex with Gq and the neuropeptide QRFP, which exhibits classic structural features in ligand recognition and receptor activation for Class A peptide GPCRs.A unique feature is that the N-terminal region of the receptor forms a helical structure to interact with the N-terminal domain of the ligand.Based on the structural analysis, the authors discussed the mechanism for ligand binding and selectivity.Overall, this is a high-quality structural study on a peptide GPCR.
Thank you very much for your evaluation of our paper.We believe that our paper was now much improved according to your suggestions.

Major issues:
1.There is no any functional data to support the structural insights.While the authors reference earlier mutagenesis studies, validating their structure through additional experiments is crucial.
If nearly all interactions between the ligand and the receptor were predicted by previous studies, the structural insights in this paper may not significantly advance the field.
According to the suggestion, we made a total of 33 mutants of amino acid residues involved in peptide binding and evaluated their G q activities by a TGFα shedding assay (Supplementary Fig. 1a-d, Supplementary Table 1).The expression levels of the mutants were accurately quantified by FACS.Mutants with significantly reduced expression were compared to the wild type, in which the plasmid amount was lowered to achieve the similar expression level.

The 'two-site' binding mechanism has been well established for many Class A peptide GPCRs
including chemokine receptors and the C5a receptor.Furthermore, the roles of the receptor Ntermini in peptide ligand engagement have been well studied by multiple biophysical methods.
Comparison with those GPCRs is important for elucidating the peptide recognition mechanism specific to GPR103.
As the reviewer pointed out, the two site binding mechanism has been proposed in other class A GPCRs.According to the suggestion, we added the structural comparison with C5aR, CXCR2, and thyroid stimulating hormone receptor (TSHR) (Fig. 7) (lines 355 to 374).TSHR is a GPCR with a large leucine-rich repeat domain at the N-terminus, but the transmembrane region is categorized as a class A GPCR, and thus we included the structural comparison.
3. The Supplementary Figure 2 indicates that the map resolution of the receptor's extracellular region, including the extracellular loops, is around 5Å after local refinement.This resolution is insufficient for precise modeling of the side chains.Therefore, it is important for the authors to show electron density maps of the critical residues of GPR103 involved in peptide binding in the Supplementary data to supporting their structural insights.
Thank you for your suggestion.We added the density map of the residues involved in peptide binding in Supplementary Fig. 3.

Other minor issues:
4. There is a discrepancy between the ligand-interacting residues shown in the main Figure 3 and those shown in the Supplementary Figure 3.The authors need to determine the accurate interacting residues and show them in the figures.
According to the suggestion, we reviewed the interacting residues and corrected the discrepancy between Fig. 3 (now Fig. 2) and Supplementary Fig. 5.Moreover, we corrected the Fig. 2 more readable.
Reviewer #2 (Remarks to the Author): In this study, Iwama et al. elucidated the structure of the QRFPR in complex with its ligand 26RFa and Gαq using cryo-EM.In the introduction, the authors present the family of RF-amide peptides and describe the peptides 43RFa and 26RFa and the corresponding receptor GPR103/QRFPR in more detail.In line 73, it is written, that the QRFPR is a Gαq-coupled receptor, however Ma et al. describe that GPR103 dually couples to Gαq andGαi/o proteins (doi: 10.1016/j.bbamcr.2021.119046).Can the authors comment on this?
We appreciate your thoughtful comments.We first performed a NanoBiT dissociation assay to confirm the activation of G q and G i/o by GPR103.The results showed activation of G o by GPR103, RFamide/RF-amide/RF amide general: better use QRFP26/43 or 26RFa/43RFa as QRFP is used for both peptides 37use names of receptors instead of GPR as ligands are known 246: including 335: In reference 38 a W6.48Q mutant is used, which doesn't really fit here