Cyclic Analogues of the Chemerin C-Terminus Mimic a Loop Conformation Essential for Activating the Chemokine-like Receptor 1

Abstract

The chemokine-like receptor 1 (CMKLR1) is a promising target for treating autoinflammatory diseases, cancer, and reproductive disorders. However, the interaction between CMKLR1 and its protein–ligand chemerin remains uncharacterized, and no drugs targeting this interaction have passed clinical trials. Here, we identify the binding mode of chemerin-9, the C-terminus of chemerin, at the receptor by combining complementary mutagenesis with structure-based modeling. Incorporating our experimental data, we present a detailed model of this binding site, including experimentally confirmed pairwise interactions for the most critical ligand residues: Chemerin-9 residue F8 binds to a hydrophobic pocket in CMKLR1 formed by the extracellular loop (ECL) 2, while F6 interacts with Y2.68, suggesting a turn-like structure. On the basis of this model, we created the first cyclic peptide with nanomolar activity, confirming the overall binding conformation. This constrained agonist mimics the loop conformation adopted by the natural ligand and can serve as a lead compound for future drug design.

Ligand-Induced and -Independent Internalization of the Chemerin Receptor GPR1

“…We previously showed that cyclic chemerin-9 derivates activate CMKLR1 with high potency, and the same holds true for GPR1 (Fig. 6) [27]. This highlights that the binding mode of the chemerin C-terminus is highly related at both receptors.…”

“…First, we determined the minimal portion of the chemerin protein needed for full arrestin recruitment, our functional readout, in order to identify the portion of chemerin that engages the receptor. In parallel, we constructed a comparative model of GPR1 to identify candidate residues in the extracellular loops and the upper portions of the transmembrane (TM) helices that can form the putative binding site for chemerin; this was done bearing in mind the previously identi ed binding pocket of chemerin at the CMKLR1 [27]. Consequently, we determined an interaction point by complementary mutagenesis, and probed the internal conformation of the minimal activation sequence employing cyclized peptides.…”

“…In CMKLR1, positions 4.67 and 4.69 consist of alanine and leucine, respectively. Exchanging these for a valine and a phenylalanine decreased the EC 50 shift between chemerin-9 and [L 8 ]-chemerin-9 by tightening the hydrophobic pocket formed by ECL2 [27]. Now, we reversed this experiment by introducing the V 4.67 A_F 4.69 L mutants into GPR1.…”

“…A Hydrophobic Pocket Formed by the ECL2 of GPR1 Contributes to Ligand Binding Chemerin-9 residue F 8 binds to a hydrophobic pocket in the ECL2 of CMKLR1 [27]. The ligand-binding residues are highly conserved between CMKLR1 and GPR1.…”

“…Homology models of GPR1 in the apo state were produced using RosettaCM [55] with the crystal structures of C5aR1 [56] (PDB: 6c1r), CCR9 [57] (PDB: 5lwe), CXCR4 [58] (PDB: 3odu), APJR [59] (PDB: 5vbl) and AT1R [60] (PDB: 4zud) as templates as described previously [27,28]. In brief, crystal structures were stripped of fusion proteins, ions, etc., followed by threading of the CMKLR1 sequence onto the template structures according to the sequence alignment given in Table S1.…”