Die Entfaltung der C‐terminalen α‐Helix des Neuropeptids Y ist entscheidend für die Bindung und Aktivierung des Y₂‐Rezeptors

Abstract

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.

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor

“…Chemie the ligand and forms with it ac luster of hydrophobic interactions,w hich were validated by mutagenesis and resemble the hydrophobic patch predicted between NPY and the ECL2 of Y 2 R( L 24 and I 28 with I 4.71 and I 4.77 ). [11] The ECL2 was also reported to form part of the ligand binding pocket of Y 1 R. [9] In general, ECL2 is known to be relevant for ligand recognition and signaling in several GPCRs. [37] Finally,crosslinking hits were observed in ECL3, although the intensity was overall lower compared to the other two regions.S till, the model predicts ah ydrophobic cluster between NPY and Y 5 R. Interestingly,E CL3 is not crucial for ligand binding at Y 2 Ra nd Y 4 R, whereas it has been reported to be important for Y 1 R. [9] Overall, the lack of crosslinking hits in deep regions of the TM helices suggests NPY achieves aq uite superficial pose within the binding pocket of Y 5 R, which is well reflected in the model.…”

“…[11] Q 3.32 is highly conserved among the YRs and this interaction likely represents ah allmark for the entire multiligand/multireceptor system. [21] With respect to the N-terminus of the ligand, our model does not predict direct interactions with the Y 5 R, similar to the NPY-Y 2 Ra nd PP-Y 4 Rm odels, [11][12][13] whereas the Nterminus of NPY was suggested to interact extensively with Y 1 R. [9] Indeed, N-terminally truncated NPY can bind Y 2 Rbut not Y 1 R. [40] Interestingly,w hile Ala substitution the Nterminus of NPY barely affected Y 5 Ra ctivation, truncation of the 12 N-terminal residues reduced the potencyo ft he ligand by 8-fold. As altering the structure of the N-terminal region of NPY can heavily affect ligand potencye ven at YR subtypes that tolerate its truncation, [41] the N-terminal segment may stabilize the overall conformation of the peptide.…”

“…[39] On the basis of our model and the mutagenesis data, we further propose that the C-terminal amide of NPY interacts with Q 3.32 in TM3 of Y 5 R, as observed in the homologous NPY-Y 2 Rm odel. [11] Q 3.32 is highly conserved among the YRs and this interaction likely represents ah allmark for the entire multiligand/multireceptor system. [21] With respect to the N-terminus of the ligand, our model does not predict direct interactions with the Y 5 R, similar to the NPY-Y 2 Ra nd PP-Y 4 Rm odels, [11][12][13] whereas the Nterminus of NPY was suggested to interact extensively with Y 1 R. [9] Indeed, N-terminally truncated NPY can bind Y 2 Rbut not Y 1 R. [40] Interestingly,w hile Ala substitution the Nterminus of NPY barely affected Y 5 Ra ctivation, truncation of the 12 N-terminal residues reduced the potencyo ft he ligand by 8-fold.…”

“…Only the structures of the Y 1 Rand most recently the Y 2 R, both in complex with smallmolecule antagonists,h ave been solved. [9,10] Computational models of peptide-YR complexes have been proposed, based on experimental data obtained with different approaches:the NPY-Y 1 R, [9] theN PY-Y 2 R, [11] and the PP-Y 4 Rc omplex. [12,13] Some differences in the binding mode of Yp eptides have been identified.…”

Binding of Natural Peptide Ligands to the Neuropeptide Y5 Receptor

“…the ligand and forms with it ac luster of hydrophobic interactions,w hich were validated by mutagenesis and resemble the hydrophobic patch predicted between NPY and the ECL2 of Y 2 R( L 24 and I 28 with I 4.71 and I 4.77 ). [11] The ECL2 was also reported to form part of the ligand binding pocket of Y 1 R. [9] In general, ECL2 is known to be relevant for ligand recognition and signaling in several GPCRs. [37] Finally,crosslinking hits were observed in ECL3, although the intensity was overall lower compared to the other two regions.S till, the model predicts ah ydrophobic cluster between NPY and Y 5 R. Interestingly,E CL3 is not crucial for ligand binding at Y 2 Ra nd Y 4 R, whereas it has been reported to be important for Y 1 R. [9] Overall, the lack of crosslinking hits in deep regions of the TM helices suggests NPY achieves aq uite superficial pose within the binding pocket of Y 5 R, which is well reflected in the model.…”