Structural Perspective on Ancient Neuropeptide Y-like System reveals Hallmark Features for Peptide Recognition and Receptor Activation

Highlights

• Many peptide–GPCR signaling systems are evolutionary conserved down to basic animals.

• We illuminate binding of three distinct neuropeptide Y-like ligands in the FLP/NPR system of C. elegans.

• We identify E^5.23(ECL2) and Q^3.32 of the receptors are family-conserved residues critical for affinity and efficacy.

• NanoBRET based ligand binding assays demonstrate flexibility of peptide ligands in the binding pocket.

• Comparing binding modes in evolutionary ancient homologs can identify key binding and activation mechanisms.

Abstract

The neuropeptide Y (NPY) family is a peptide-activated G protein-coupled receptor system conserved across all bilaterians, and is involved in food intake, learning, and behavior. We hypothesized that comparing the NPY system in evolutionarily ancient organisms can reveal structural determinants of peptide recognition and receptor activation conserved in evolution. To test this hypothesis, we investigated the homologous FLP/NPR system of the protostome C. elegans. For three prototypic peptide–receptor complexes representing different ligand types, we integrate extensive functional data into structural models of the receptors. Common features include acidic patches in the extracellular loops (ECLs) of the receptors that cooperatively ‘draw’ the peptide into the binding pocket, which was functionally validated in vivo. A structurally conserved glutamate in the ECL2 anchors the peptides by a conserved salt bridge to the arginine of the RFamide motif. Beyond this conserved interaction, peptide binding show variability enabled by receptor-specific interactions. The family-conserved residue Q^3.32 is a key player for peptide binding and receptor activation. Altered interaction patterns at Q^3.32 may drastically increase the efficacy to activate the receptor.

Introduction

A substantial portion of G protein-coupled receptors (GPCRs) is activated by endogenous peptide or protein ligands. These peptide and protein GPCRs are involved in many physiological processes and are important pharmacological targets. Understanding how peptides interact with their receptors is an essential step towards the development of more potent and selective probe molecules that can validate these receptors as targets and ultimately spur therapeutic development. Specifically, the recognition of the large and flexible peptides by the receptor and the mechanisms for receptor-subtype selectivity are intensively studied.

Reflecting their essential physiological functions, many peptide GPCR systems are conserved down to basic animals, which opens up avenues to study conserved aspects of peptide–receptor interactions, and a potential co-evolution of the binding pockets. Among several other systems, the neuropeptide Y (NPY) system has been present already in the common ancestor of protostomes and deuterostomes, the ‘ur-bilaterian’,1, 2 and is thus expected to have homologs in all bilaterians. In humans, the NPY family has an essential role in regulating food intake and is intensively studied as a potential target for modulating food consumption in the context of obesity.3, 4, 5 Additional functions of the NPY system include the regulation of memory retention, mood and anxiety.[6], 7, 8, 9 NPY and the related peptides PYY and PP activate four cognate GPCRs (Y₁, Y₂, Y₄ and Y₅) in humans. The peptides feature a C-terminal arginine-phenylalanine/tyrosine sequence with an amidated carboxyl-terminus (–RxRF/Yamide), which is essential for their activity. High-resolution structural insights of NPY binding to its cognate receptors are currently lacking, but structural models suggest that the arginine residues of this sequence contact a conserved D^6.59 on the top of transmembrane helix 6 (TM6) of the receptors, and that the C terminus of the peptide accommodates a binding pocket in the TM bundle.10, 11, 12

Recently, we showed that the FMRFamide-like peptide (FLP)/neuropeptide receptor-resemblence (NPR) system of the protostome Caenorhabditis elegans is, beyond sequence similarities, pharmacologically highly similar to the human NPY system.¹³ Most notably, the peptide ligands feature cross-species activity, and human NPY receptors can functionally replace C. elegans npr-1 null mutants in an in vivo context.¹³ The FLP/NPR system in C. elegans is expanded and comprises more than 70 FLP ligands (incl. isoforms originating from local genome duplications) and 40 NPR receptors14, 15 with apparently redundant activation profiles.¹³ Interestingly, many of the short peptides only contain a minimal C-terminally conserved –ϕRFamide (ϕ = hydrophobic) sequence, and lack a second Arg/Lys residue at the -4 position which still suffices for receptor activation. To facilitate comparison to human NPY-like peptides, positions are counted from the C terminus. FLP-21 (GLGPRPLRFamide) activates several NPRs. It features nanomolar potencies for NPR-1 and NPR-11, and also activates the human Y₂ and Y₄ receptor with sub-micromolar potency.¹³ This peptide carries a second arginine residue at the -5 position (one position displaced from the ‘classic’ -4 position of NPY). Recently, three FLPs with increased length and a ‘classic’ C-terminal –RxRF/Yamide have been functionally characterized (FLP-27, FLP-33, FLP-34), which act at NPR-11 and human NPY receptors,¹³ but have limited activity at other NPRs.¹³ Isoform 1 of FLP-34 (FLP-34-1: ADISTFASAINNAGRLRYamide), activates NPR-11 with nanomolar potency.

Here, we illuminate how FLP-21 and FLP-34-1 bind NPR-1 and NPR-11 to resolve the contribution of the C-terminal –RFamide and –RxRYamide motif, respectively, for receptor selectivity and the activation process. For each of the three peptide–receptor combinations (FLP-21 – NPR-1; FLP-21 – NPR-11; FLP-34-1 – NPR-11), we integrate experimental restraints into a comparative model, and dock the peptides with full flexibility, which converged into well-defined binding poses. We demonstrate that binding of the FLP ligands is heavily dependent on the conserved C-terminal motifs, which involves a salt bridge of the arginine at the -2 position (RFamide) to a conserved acidic receptor residue in the ECL2. Additional acidic clusters aid the guidance of the peptides into the binding pocket. Moreover, the C-terminal Phe/Tyr-amide is required for both, affinity to the receptor and receptor activation. The shape of the binding pocket and the receptor residues involved in peptide binding are conserved to a high degree in the human NPY receptors, suggesting a similar mode of action. Mutation of the family-wide conserved Q^3.32 in the transmembrane binding pocket of NPR-11 leads to an altered binding mode of FLP-21 at this receptor, which increases G protein signaling, thus identifying interactions for efficacious receptor activation.