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Identification of the conserved Asp 6. 59 residue in the hPrRPR sequence as potential point of interaction. A, conservation of Asp 6.59 shown in the amino acid sequence alignment. The region of upper TMH6 and the beginning of the subsequent EL3 of the four human Y receptor subtypes and the PrRPR are presented. B, comparison of the C-terminal amino acids of the Y receptor ligands and the PrRP20. C, snake plot representing the sequence of the human PrRPR. Residues highlighted in black were investigated as double mutants in the D6.59R construct. Selective alanine scanning was performed on residues pictured in gray, resulting in no functional alteration. Residues with white letters on gray correspond to the X.50 nomenclature (16). D, IP accumulating signal transduction assay performed for 1 h with COS-7 cells in a concentration-response dependent manner reveals an impact of D6.59A PrRPR in comparison with the WT PrRP receptor. Data represent the mean S.E. of multiple independent experiments (n 32 for hPrRPR and n 12 for D6.59A PrRPR). Receptor activity is expressed as percentage of the full response of PrRP20 at the WT PrRP receptor.
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The prolactin-releasing peptide receptor and its bioactive RF-amide peptide (PrRP20) have been investigated to explore the
ligand binding mode of peptide G-protein-coupled receptors (GPCRs). By receptor mutagenesis, we identified the conserved aspartate
in the upper transmembrane helix 6 (Asp6.59) of the receptor as the first position that directly...
Citations
... Our mutagenesis analyses showed that D/E 6.59 is critical for the activities of NPVF and NPFF and also has a moderate impact on PrRP activity (Fig. 5d-f; Supplementary Fig. S3c and Tables S3-S5). These results are consistent with previous findings that D 6.59 is important for the activation potency of NPFF1/2R 38 and PrPR 39 . In contrast, mutating D 6.59 to alanine almost did not affect 26RFa activity (Fig. 5c). ...
... These findings highlight the significance of the hydrophobic interaction 230 between Phe26 and F289 6.51 for the activity of 26RFa and also suggest a potential role for V 3.36 and 231 L 6.51 in the peptide activity towards other RF-amide receptors. Q123 3.32 and Q318 7. 39 surrounding 232 the amide group form intermolecular hydrogen bonds with the RF-amide segment in 26RFa, which 233 are essential for the peptide's activity (Fig. 3c, d). Sequence alignment shows that the glutamine at 234 position 3.32 is conserved, whereas glutamine at position 7.39 in QRFPR is substituted with 235 histidine in other RF-amide receptors (Fig. 5b). ...
... In the case of KP-10, Q 3.32 also exhibits a 237 remarkable impact. Differently, H 7. 39 shows only minor effects on its activity (Fig. 5g). 238 239 Collectively, in contrast to KP-10, the RF-amide segment is more crucial for the activity of other 240 RF-amide peptides. ...
The neuropeptide 26RFa, a member of the RF-amide peptide family, activates the pyroglutamylated RF-amide peptide receptor (QRFPR), a class A GPCR. The 26RFa/QRFPR system plays critical roles in energy homeostasis, making QRFPR an attractive drug target for treating obesity, diabetes, and eating disorders. However, the lack of structural information has hindered our understanding of the peptide recognition and regulatory mechanism of QRFPR, impeding drug design efforts. In this study, we determined the cryo-EM structure of the Gq-coupled QRFPR bound to 26RFa. The structure reveals a unique assembly mode of the receptor extracellular regions and the peptide N-terminus and elucidates the recognition mechanism of the C-terminal heptapeptide of 26RFa within the transmembrane binding pocket of QRFPR. The study also clarifies the similarities and distinctions in the binding pattern of the RF-amide moiety in five RF-amide peptides and the RY-amide segment in neuropeptide Y. These findings deepen our understanding of the RF-amide peptides recognition, aiding in the rational design of drugs targeting QRFPR and other RF-amide receptors.
... For instance, a ligand may lose its activity when a Lys/Arg residue is mutated to Glu. If the activity is recovered when a Glu/Asp residue in the receptor is mutated to Lys/Arg, an intermolecular ionic interaction between the two positions can well be hypothesized [54]. However, these drastic alterations of the local environment of the receptor and peptide often provoke artificial conformational changes and render the receptor and/or ligand inactive. ...
Many biological functions of peptides are mediated through G protein-coupled receptors (GPCRs). Upon ligand binding, GPCRs undergo conformational changes that facilitate the binding and activation of multiple effectors. GPCRs regulate nearly all physiological processes and are a favorite pharmacological target. In particular, drugs are sought after that elicit the recruitment of selected effectors only (biased ligands). Understanding how ligands bind to GPCRs and which conformational changes they induce is a fundamental step toward the development of more efficient and specific drugs. Moreover, it is emerging that the dynamic of the ligand–receptor interaction contributes to the specificity of both ligand recognition and effector recruitment, an aspect that is missing in structural snapshots from crystallography. We describe here biochemical and biophysical techniques to address ligand–receptor interactions in their structural and dynamic aspects, which include mutagenesis, crosslinking, spectroscopic techniques, and mass-spectrometry profiling. With a main focus on peptide receptors, we present methods to unveil the ligand–receptor contact interface and methods that address conformational changes both in the ligand and the GPCR. The presented studies highlight a wide structural heterogeneity among peptide receptors, reveal distinct structural changes occurring during ligand binding and a surprisingly high dynamics of the ligand–GPCR complexes.
... C-end (Boyle, et al., 2005;Findeisen, et al., 2011;Rathmann, et al., 2012 ...
... Another conserved residue in the RF-amide receptors is aspartate 6.59, mutation of this residue significantly attenuates peptide activity (Findeisen, et al., 2011). From functional and modelling studies, it is suggested that aspartate 6.59 interacts with the arginine of the peptide RF-amide motif (Rathmann, et al., 2012). ...
Many physiological processes are controlled through the activation of G protein-coupled receptors (GPCRs) by regulatory peptides, making peptide GPCRs particularly useful targets for major human diseases such as diabetes and cancer. Peptide GPCRs are also being evaluated as next-generation targets for the development of novel anti-parasite agents and insecticides in veterinary medicine and agriculture. Resolution of crystal structures for several peptide GPCRs has advanced our understanding of peptide-receptor interactions and fuelled interest in correlating peptide heterogeneity with receptor binding properties. In this review, the knowledge of recently crystalized peptide-GPCR complexes, previously accumulated peptide structure-activity relationship (SAR) studies, receptor mutagenesis and sequence alignment are integrated to better understand peptide binding to the transmembrane cavity of Class A GPCRs. Using SAR data, we show that peptide Class A GPCRs can be divided into groups with distinct hydrophilic residues. These characteristic residues help explain the preference of a receptor to bind the C-terminal free carboxyl group, the C-terminal amidated group, or the N-terminal ammonium group of peptides. SIGNIFICANCE STATEMENT: The recent reporting of crystal structures of GPCRs bound to peptides allows structural diversity of peptide binding to be assessed for the first time. Furthermore, the structural data facilitates interpretation of peptide SAR studies and allows extrapolation of findings to related GPCRs. Thus, our polar residue analysis enables to group the peptide receptors highlighting conserved residues important for peptide binding. Despite a large heterogeneity in possible binding modes of peptides within GPCRs, some level of generalization of peptide binding can be established through the analysis of the peptide terminal end binding to the transmembrane helical cavity of GPCRs.
G protein-coupled receptors (GPCRs) represent the largest membrane protein family anda significant target class for therapeutics. Receptors from GPCRs’ largest class, class A, influencevirtually every aspect of human physiology. About 45% of the members of this family endogenouslybind flexible peptides or peptides segments within larger protein ligands. While many of thesepeptides have been structurally characterized in their solution state, the few studies of peptides intheir receptor-bound state suggest that these peptides interact with a shared set of residues andundergo significant conformational changes. For the purpose of understanding binding dynamicsand the development of peptidomimetic drug compounds, further studies should investigate thepeptide ligands that are complexed to their cognate receptor.
Anorexigenic peptides offer promise as potential therapies targeting the escalating global obesity epidemic. Prolactin-releasing peptide (PrRP), a novel member of the RFamide family secreted by the hypothalamus, shows therapeutic potential by decreasing food intake and body weight in rodent models via GPR10 activation. Here we describe the design of a long-acting PrRP using our recently developed novel multiple ethylene glycol-fatty acid (MEG-FA) stapling platform. By incorporating serum albumin binding fatty acids onto a covalent side chain staple, we have generated a series of MEG-FA stapled PrRP analogs with enhanced serum stability and in vivo half-life. Our lead compound 18-S4 exhibits good in vitro potency and selectivity against GPR10, improved serum stability and extended in vivo half-life (7.8 h) in mouse. Furthermore, 18-S4 demonstrates a potent body weight reduction effect in a diet-induced obesity (DIO) mouse model, representing a promising long-acting PrRP analog for further evaluation in the chronic obesity setting.
Structural characterization of the human Y4 receptor (hY4R) interaction with human pancreatic polypeptide (hPP) is crucial, not only for understanding its biological function but
also for testing treatment strategies for obesity that target this interaction. Here, the interaction of receptor mutants
with pancreatic polypeptide analogs was studied through double-cycle mutagenesis. To guide mutagenesis and interpret results,
a three-dimensional comparative model of the hY4R-hPP complex was constructed based on all available class A G protein-coupled receptor crystal structures and refined using
experimental data. Our study reveals that residues of the hPP and the hY4R form a complex network consisting of ionic interactions, hydrophobic interactions, and hydrogen binding. Residues Tyr2.64, Asp2.68, Asn6.55, Asn7.32, and Phe7.35 of Y4R are found to be important in receptor activation by hPP. Specifically, Tyr2.64 interacts with Tyr27 of hPP through hydrophobic contacts. Asn7.32 is affected by modifications on position Arg33 of hPP, suggesting a hydrogen bond between these two residues. Likewise, we find that Phe7.35 is affected by modifications of hPP at positions 33 and 36, indicating interactions between these three amino acids. Taken
together, we demonstrate that the top of transmembrane helix 2 (TM2) and the top of transmembrane helices 6 and 7 (TM6–TM7)
form the core of the peptide binding pocket. These findings will contribute to the rational design of ligands that bind the
receptor more effectively to produce an enhanced agonistic or antagonistic effect.
Structure-based drug design is frequently used to accelerate the development of small-molecule therapeutics. Although substantial progress has been made in X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, the availability of high-resolution structures is limited owing to the frequent inability to crystallize or obtain sufficient NMR restraints for large or flexible proteins. Computational methods can be used to both predict unknown protein structures and model ligand interactions when experimental data are unavailable. This paper describes a comprehensive and detailed protocol using the Rosetta modeling suite to dock small-molecule ligands into comparative models. In the protocol presented here, we review the comparative modeling process, including sequence alignment, threading and loop building. Next, we cover docking a small-molecule ligand into the protein comparative model. In addition, we discuss criteria that can improve ligand docking into comparative models. Finally, and importantly, we present a strategy for assessing model quality. The entire protocol is presented on a single example selected solely for didactic purposes. The results are therefore not representative and do not replace benchmarks published elsewhere. We also provide an additional tutorial so that the user can gain hands-on experience in using Rosetta. The protocol should take 5-7 h, with additional time allocated for computer generation of models.
Mutagenic investigations of expressed membrane proteins are routine but the variety of modifications is limited, by the 20 canonical amino. We describe an easy and effective cysteine substitution mutagenesis method to modify and investigate distinct amino acids in vitro. The approach combines the substituted-cysteine accessibility method (SCAM) with a functional signal transduction read-out system using different thiol-specific reagents. We applied this approach to the prolactin releasing peptide receptor (PrRPR) to facilitate biochemical structure activity relationship studies of eight crucial positions. Especially for D(6.59)C, the treatment with the positively charged methanethiosulfonate (MTS)-ethylammonium led to an induced basal activity, whereas the coupling of the negatively charged MTS-ethylsulfonate almost reconstituted full activity, obviously by mimicking the wild type charged side chain. At E(5.26)C, W(5.28)C, Y(5.38)C, and Q(7.35)C accessibility was observed but hindered transfer into the active receptor conformation. Accordingly, the combination of SCAM and signaling assay is feasible and can be adapted to other GPCRs. This method circumvents the laborious way of inserting non-proteinogenic amino acids to investigate activity and ligand binding, as rising numbers of MTS-reagents allow selective side chain modification. This method pinpoints to residues, being accessible but also presents potential molecular positions to investigate the global conformation.
