Despite recent breakthroughs in G protein-coupled receptor (GPCR) structure characterization, there is only sparse data on how GPCRs recognize larger peptide ligands. Here, we integrate nuclear magnetic resonance (NMR) spectroscopy, molecular modeling, and double-cycle mutagenesis studies to obtain a structural model of the peptide hormone neuropeptide Y (NPY), bound to its human G protein-coupled Y2 receptor (Y2R). Solid-state NMR measurements of specifically isotope-labeled NPY in complex with in vitro folded Y2R reconstituted into phospholipid bicelles provide the bioactive structure of the peptide. Guided by solution NMR experiments, we find that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C-terminal α-helix of NPY, which is formed in membrane environment in the absence of the receptor, is unwound starting at T32 to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y2R.

Chemerin is an immunomodulating factor secreted predominantly by adipose tissue and skin. Processed by a variety of proteases linked to inflammation, it activates the G-protein coupled receptor chemokine-like receptor 1 (CMKLR1) and induces chemotaxis in natural killer cells, macrophages, and immature dendritic cells. Recent developments revealed the role of the nonsignaling chemerin receptor C-C chemokine receptor-like 2 (CCRL2) in inflammation. Besides further research establishing its link to inflammatory skin conditions such as psoriasis, functions in healthy skin have also been reported. Here, the current understanding of chemerin processing, signaling and physiological function has been summarized, focusing on the regulation of its activity, its different receptors and its controversially discussed role in diseases.

The excessive accumulation of adipose tissue in obesity is associated with multiple inflammatory dermatological diseases. Chemerin, a chemoattractant adipokine, dependent on proteolytical activation, is highly expressed in skin. Different proteases have been reported to activate prochemerin, but none is inherently expressed in human skin. In the present study, we identified a tissue-specific protease and investigated the underlying mechanism of activation at the molecular level. We characterized human KLK7 (kallikrein 7) as a prochemerin processing protease in vitro converting prochemerin into active chemerinF(156). The activating truncation by the protease might trigger a structural rearrangement leading to an increased affinity of chemerin to CMKLR1 (chemokine-like receptor 1). Molecular modelling and experimental data suggest an underlying ionic interaction in prochemerin C-terminal domains. These findings provide a general molecular basis for the necessity of C-terminal processing of prochemerin. Moreover, immunohistochemistry was used to investigate prochemerin, KLK7 and the recently identified KLK7 inhibitor vaspin expression in human skin biopsies, and distinct co-localization in psoriatic biopsies was observed. On the basis of these results, it is hypothesized that KLK7 activity may contribute to the development of psoriatic lesions as a consequence of excessive chemerin activation and impaired protease activity regulation by vaspin. Therefore this interaction represents an interesting target for psoriasis therapy and treatment of other obesity-related diseases.

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.

The neuropeptide Y receptor (Y R) is involved in various pathophysiological processes such as epilepsy, mood disorders, angiogenesis, and tumor growth. Therefore, the Y R is an interesting target for drug development. A detailed understanding of the binding pocket could facilitate the development of highly selective antagonists to study the role of Y R in vitro and in vivo. In this study, several residues crucial to the interaction of BIIE0246 and SF-11 derivatives with Y R were investigated by signal transduction assays. Using the experimental results as constraints, the antagonists were docked into a comparative structural model of the Y R. Despite differences in size and structure, all three antagonists display a similar binding site, including a deep hydrophobic cavity formed by transmembrane helices (TM) 4, 5, and 6, as well as a hydrophobic patch at the top of TM2 and 7. Additionally, we suggest that the antagonists block Q , a position that has been shown to be crucial for binding of the amidated C terminus of NPY and thus for receptor activation.

Trotz jüngster Fortschritte in der Strukturbestimmung von G-Protein-gekoppelten Rezeptoren (GPCR) gibt es nur wenige Daten zur Interaktion von GPCRs mit grçßeren Peptidliganden. In dieser Arbeit kombinieren wir Methoden der NMR-Spektroskopie,d er molekularen Modellierung und der ortsgerichteten komplementären Mutagenese und präsentieren ein Strukturmodell des Peptidhormons Neuropeptid Y (NPY) im Komplex mit seinem humanen G-Protein-gekoppelten Y 2 -Rezeptor (Y 2 R). Festkçrper-NMR-Messungen von spezifisch isotopenmarkiertem NPY im Komplex mit in vitro gefaltetem und in Phospholipid-Bizellen rekonstituiertem Y 2 R liefern Daten zur bioaktiven Peptidstruktur.U nterstützt von Lçsungs-NMR-Studien konnten Interaktionen des Liganden mit dem zweiten extrazellulären Loop des Rezeptors über hydrophobe Kontakte nachgewiesen werden. Die C-terminale a-Helix von NPY,d ie sich in einer Membranumgebung in Abwesenheit des Rezeptors bildet, entfaltet sich ab T 32 ,u m einen optimalen Kontakt tief in der Bindungstasche des transmembranen Bereichs des Y 2 Rzue rmçglichen.

No abstract

Chemerin is a small chemotactic protein and a modulator of the innate immune system. Its activity is mainly mediated by the chemokine-like receptor 1 (CMKLR1), a receptor expressed by natural killer cells, dendritic cells, and macrophages. Downregulation of chemerin is part of the immune evasion strategy exploited by several cancer types, including melanoma, breast cancer, and hepatocellular carcinoma. Administration of chemerin can potentially counteract these effects, but synthetically accessible, metabolically stable analogs are required. Other tumors display overexpression of CMKLR1, offering a potential entry point for targeted delivery of chemotherapeutics. Here, we present cyclic derivatives of the chemerin C-terminus (chemerin-9), the minimal activation sequence of chemerin. Chemerin-9 derivatives that were cyclized through positions four and nine retained activity while displaying full stability in blood plasma for more than 24 h. Therefore, these peptides could be used as a drug shuttle system to target cancer cells as demonstrated here by methotrexate conjugates.