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Structural basis of G protein–Coupled receptor CMKLR1 activation and signaling induced by a chemerin-derived agonist [1]

['Xuan Zhang', 'Department Of Pharmacology', 'Chemical Biology', 'School Of Medicine', 'University Of Pittsburgh', 'Pittsburgh', 'Pennsylvania', 'United States Of America', 'Tina Weiß', 'Institute Of Biochemistry']

Date: 2023-12

Chemokine-like receptor 1 (CMKLR1), also known as chemerin receptor 23 (ChemR23) or chemerin receptor 1, is a chemoattractant G protein–coupled receptor (GPCR) that responds to the adipokine chemerin and is highly expressed in innate immune cells, including macrophages and neutrophils. The signaling pathways of CMKLR1 can lead to both pro- and anti-inflammatory effects depending on the ligands and physiological contexts. To understand the molecular mechanisms of CMKLR1 signaling, we determined a high-resolution cryo-electron microscopy (cryo-EM) structure of the CMKLR1-G i signaling complex with chemerin9, a nanopeptide agonist derived from chemerin, which induced complex phenotypic changes of macrophages in our assays. The cryo-EM structure, together with molecular dynamics simulations and mutagenesis studies, revealed the molecular basis of CMKLR1 signaling by elucidating the interactions at the ligand-binding pocket and the agonist-induced conformational changes. Our results are expected to facilitate the development of small molecule CMKLR1 agonists that mimic the action of chemerin9 to promote the resolution of inflammation.

Funding: Grants from the National Institutes of Health (NIH) in the US R35GM128641 (to C.Z.), R01CA255250 (to M.F.), R01CA258778 (to M.F.), and R01GM139297 (to I.B.). The German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) project number 209933838, CRC1052/3, C08 (to A.G.B-S.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: The 3D cryo-EM density map of CMKLR1-Gi-scFv16 complex with chemerin9 has been deposited in the Electron Microscopy Data Bank under the accession numbers EMD-40450. Atomic coordinates for the atomic model have been deposited in the Protein Data Bank (PDB) under the accession numbers 8SG1. The FACS data has been deposited to FlowRepository with the ID FR-FCM-Z6US ( http://flowrepository.org/id/FR-FCM-Z6US ).

Copyright: © 2023 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

To address those questions in CMKLR1 physiology, pharmacology, and drug development, we mainly focused on the peptide agonists and solved a cryo-electron microscopy (cryo-EM) structure of the CMKLR1-G i signaling complex with chemerin9, which, in our assays, induced a phenotype of macrophages that does not fall into the oversimplified M1- nor M2-macrophages paradigm [ 32 ]. Together with molecular dynamics (MD) simulations and mutagenesis studies, our structure revealed critical molecular features for the binding of chemerin9 and shed light on the molecular mechanism by which chemerin9 activates CMKLR1 to promote G i signaling. The structural information is anticipated to facilitate the development of synthetic small molecule agonists mimicking the action of chemerin9 for CMKLR1.

The potential roles of CMKLR1 signaling in inflammation may provide promising new opportunities for developing anti-inflammatory drugs. However, although there are plenty of peptide agonists derived from chemerin, the limited availability of synthetic ligands of CMKLR1 has impeded pharmacological investigation and drug development in this area. One potent antagonist of CMKLR1 named CCX832 has been identified, but the chemical structure remains undisclosed. Additionally, a few 2-aminobenzoxazole analogues have been reported as CMKLR1 inhibitors [ 28 , 29 ]. Nevertheless, the only commercially available small-molecule CMKLR1 antagonist is α-NETA, which can target other proteins and shows low micromolar potency against CMKLR1 [ 30 ]. As to CMKLR1 agonists, several chemerin-derived peptide agonists have been developed [ 1 , 7 , 9 ], and one CMKLR1 antibody that functions as a receptor agonist was reported to promote inflammation resolution in chronic colitis models [ 31 ]. Yet, no small-molecule agonists of CMKLR1 have been reported so far. In contrast, numerous small-molecule agonists have been developed for the closely related pro-resolving GPCR, FPR2, as novel anti-inflammatory agents for clinical investigation [ 12 ].

Chemerin9 has been tested and shown positive therapeutic effects in several animal disease models of cardiovascular diseases [ 16 , 17 ], memory impairment [ 18 ], and diabetes [ 19 ]. Interestingly, recent studies provided strong evidence indicating that the activation of CMKLR1 mediates the protective effects of ω3-polyunsaturated fatty acids (PUFAs) in aortic valve stenosis 18 , atherosclerosis 19 , pulmonary hypertension [ 20 ], and depression [ 21 , 22 ], which involve resolvin E1 (RvE1) [ 23 , 24 ]. RvE1 is a specialized pro-resolvin lipid mediator (SPM) that has been suggested to act on CMKLR1 to promote the resolution of inflammation [ 25 – 27 ]. However, it is not clear whether RvE1 can directly bind to and activate CMKLR1 to induce G i signaling.

The apparent contradictory functional roles of CMKLR1 in inflammation may be associated with the spatiotemporal regulation of the inflammation processes in cells. It is possible that in the early stage of inflammation, the CMKLR1 signaling is mainly pro-inflammatory by promoting chemotaxis and activation of DCs and macrophages, whereas at the late stage of inflammation, CMKLR1 activation can induce pro-resolving pathways to dampen inflammation. Another possibility for the differing effects of CMKLR1 signaling on inflammation is that different chemerin isoforms may activate CMKLR1 to induce distinct signaling outcomes [ 1 ]. Indeed, 2 synthetic peptides, chemerin9 [ 13 , 14 ] and chemerin15 [ 15 ] corresponding to the 149–157 and the 141–155 amino acid segments of chemerin, respectively, have been characterized as stable agonists of CMKLR1 that induce anti-inflammatory effects [ 9 ].

Chemerin is usually considered as an adipocytokine or adipokine since it is produced by adipocytes and can act on CMKLR1 to regulate adipogenesis and energy metabolism in adipose tissue [ 1 , 4 , 7 , 8 ]. Increased levels of chemerin and CMKLR1 have been linked to obesity and insulin resistance [ 3 , 4 , 8 ]. In addition, the chemerin-CMKLR1 signaling axis also plays pleiotropic roles in inflammation. Chemerin can act as a potent chemoattractant to enhance chemotaxis of monocytes especially dendritic cells (DCs) through CMKLR1 signaling [ 4 , 7 , 9 , 10 ]. In this respect, the activation of CMKLR1 promotes the onset of inflammation. On the other hand, numerous studies suggested that the signaling of CMKLR1 could also resolve inflammation [ 1 , 7 , 9 ]. Such multifaceted functional roles in inflammation are also observed for another chemoattractant GPCR, the formyl peptide receptor 2 (FPR2) [ 11 , 12 ], which is closely related to CMKLR1.

Chemerin is a 163-amino acid preprotein encoded by the retinoic acid receptor responder protein 2 (RARRES 2) gene that is up-regulated by the retinoid drug tazarotene [ 1 – 4 ]. Cleavage of the N-terminal 20-amino acid signal peptide and the C-terminal 6-amino acid segment leads to the most active form of chemerin containing residues 21–157, which functions as the endogenous ligand to activate the chemokine-like receptor 1 (CMKLR1) or chemerin receptor 23 (ChemR23) or chemerin receptor 1 [ 2 – 5 ]. Other isoforms of chemerin due to cleavage at different sites of the C-terminal region have also been detected in vivo with lower potencies in activating CMKLR1 compared to the 21–157 isoform [ 1 ]. CMKLR1 belongs to a family of G i -coupled chemoattractant G protein–coupled receptors (GPCRs) in the γ-subgroup of the Class A GPCRs [ 6 ]. Other members of this family as close phylogenetic neighbors of CMKLR1 include receptors for anaphylatoxin C5a, formyl peptides, and prostaglandin D2 (PGD 2 ) [ 6 ].

Results

Complex phenotypic changes of macrophages induced by chemerin9 Despite the positive therapeutic effects of chemein9 in multiple disease models, it remains unclear whether chemerin9 can induce an anti-inflammatory phenotype of macrophages. To investigate the functional effects of the chemerin9-CMKLR1 signal axis in macrophages, we treated primary human macrophages with chemerin9, along with IFNg and LPS as the reference signal molecules to induce the M1-like macrophages and IL-10 as the reference signal molecule to induce the M2-like macrophages. We then checked the expression levels of 4 cell surface proteins, HLA-DR, CD206, CD163, and CD86 (Figs 1A and S1), which are characteristic markers of different phenotypes of macrophages [33,34]. HLA-DR is a class II major histocompatibility complex cell surface molecule, a signature cell marker for antigen-presenting cells (APCs), while CD86 is a selective ligand for the costimulatory molecule CD28, the activation of which is indispensable for optimal T cell prime and activation; together, HLA-DR and CD86 are signature cell surface markers for the canonical M1-like macrophages [34]. On the other hand, CD206, the mannose receptor (MR), is a glycosylated surface protein belonging to the scavenger receptor family. Similarly, CD163, another member of the scavenger receptor family, is the receptor for hemoglobin-haptoglobin complex. The up-regulation of CD206 and CD163 in macrophages has been associated with the anti-inflammatory and tissue-repair phenotype [33,35]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Chemerin9-induced phenotypic changes of macrophage and overall structure of the CMKLR1-G i -scFv16 complex with chemerin9. (a) The MFI fold-change of HLA-DR, CD86, CD206, and CD163 in primary macrophages under various stimulation conditions, normalized to the M0 condition (medium). Two-way ANOVA with Dunnett’s multiple comparisons test, with M0 group as control reference. n = 4, each dot represents macrophages from an independent donor. ns, not significant, *, p < 0.05, **, p < 0.01, ****, p < 0.0001 The underlying data for Fig 1A can be found in S1 Data. (b) The left and right panels show the cryo-EM density map and the overall structure, respectively. CMKLR1 is colored in blue. Chemerin9 is colored in orange. Gαi, Gβ, and Gγ subunits are colored in cyan, salmon, and dark yellow, respectively. ScFv16 is colored in grey. CMKLR1, chemokine-like receptor 1; cryo-EM, cryo-electron microscopy; MFI, mean fluorescence intensity. https://doi.org/10.1371/journal.pbio.3002188.g001 In our assays, IFNg plus LPS treatment led to elevated expression of HLA-DR and CD86 and lowered the expression of CD206 and CD163 in macrophages (Fig 1A), suggesting the pro-inflammatory M1-like phenotype [32]. IL-10 treatment induced the down-regulation of HLA-DR and CD86 and the up-regulation of CD206 and CD163 in primary human macrophages (Fig 1A), consistent with an anti-inflammatory phenotype, although the changes were statistically insignificant. When stimulated by chemerin9, the primary human macrophages showed significantly decreased levels of CD206 and CD163 as done by IFNg plus LPS, which induced inflammation (Fig 1A). However, chemerin9 did not induce the up-regulation of HLA-DR or CD86, which is a signature feature of the pro-inflammatory macrophages. In fact, we observed slightly lowered levels of these 2 markers upon chemerin9 stimulation compared to those induced by IL-10, consistent with an anti-inflammatory effect (Fig 1A). Taken together, the results suggest that the chemerin9-CMKLR1 signaling axis induced a phenotype of macrophages that does not simply fall into the oversimplified classification of the M1- and the M2-macrophages paradigm. How CMKLR1 regulates inflammation via action on macrophages needs further investigation.

Conformational dynamics of the chemerin9-CMKLR1-G i complex Chemerin9 is a potent peptide agonist of CMKLR1. To better understand the conformational dynamics of the receptor and chemerin9 in G i coupling, we performed 5 different sets of MD simulations of the CMKLR1-G i complex or CMKLR in the absence or presence of chemerin9 (see details in Methods). Each system was subjected to 3 runs of 100-ns MD simulations, resulting in a total of 1.5 microseconds of simulation data. The CMKLR1-G i complex remained stably associated throughout all simulation runs with or without chemerin9. Noticeably, we observed that CMKLR1 exhibited slightly more conformational fluctuations in the absence of chemerin9, despite being coupled and stabilized by G i (S6A Fig). These results suggest that chemerin9 may further stabilize the active conformation of CMKLR1 even in the presence of G i . As mentioned above, in our cryo-EM structure, the carboxylate group of Ser157 is located in a positively charged environment (Fig 4B). To investigate the impact of charges of chemerin9 on ligand binding, we conducted simulations of CMKLR1 bound to chemerin9 with both neutral and charged amino- and carboxyl-termini (N-ter and C-ter). Both charged and noncharged chemerin9 remained stably bound in the binding pocket throughout the simulations, indicating that the binding is overall stabilized by a network of interactions rather than specific pairs. Yet, to further evaluate the binding energy of chemerin9, we used the Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method [58], as per previous protocol [59] (Fig 6A). Our calculations showed that charged chemerin9 exhibited a lower ΔH MM/GBSA of binding, indicating a higher affinity for CMKLR1 compared to noncharged chemerin9 (Fig 6A). We attributed this increase in binding affinity primarily to the charged carboxyl termini of chemerin9. This is consistent with our mutagenesis studies suggesting the important role of charge–charge interactions between the C-terminal carboxylate group of chemerin9 and CMKLR1. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Binding of chemerin9 probed by MD simulations. (a) MM/GBSA calculations (multiple runs of 100 ns each) indicated the more favorable interactions achieved in the presence of charged amino acids at the C- and N-termini of chemerin9 (lower panel), compared to those in the presence of neutral amino acids (upper panel). (b) Gi-bound receptor exhibits closer association with chemerin9, evidenced by the closer distance between Phe156 (chemerin9) and W2736.48 (CMKLR1) observed in the runs conducted with (cyan histogram) and without (orange histogram) Gi protein bound to the receptor. The underlying data for Fig 6A and 6B can be found in S4 Data. CMKLR1, chemokine-like receptor 1; MD, molecular dynamics; MM/GBSA, Molecular Mechanics/Generalized Born Surface Area. https://doi.org/10.1371/journal.pbio.3002188.g006 During our simulations of CMKLR1 alone with charged chemerin9, we observed chemerin9 undergoing up and down movements within the binding pocket. However, in the presence of G i , which stabilized the active conformation of CMKLR1, chemerin9 gradually inserted into the pocket and moved down towards W2736.48 (Fig 6B). This observation suggests that the active CMKLR1 conformation favors a close distance between the peptide agonist and the transmission switch motif. It is possible that this state only represents a local minimum energy state, which was not captured by the cryo-EM structure. These findings offer new insights into how the coupled conformational dynamics of the receptor and the peptide agonist underlie receptor activation and signaling.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3002188

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