Binding conformation and determinants of a single-chain peptide antagonist at the relaxin-3 receptor RXFP3

Linda M Haugaard-Kedström,Han Siean Lee,Maryon V Jones,Angela Song,Vishaal Rathod,Mohammed Akhter Hossain,Ross A.D Bathgate,K Johan Rosengren

doi:10.1074/jbc.ra118.002611

Abstract

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. A linear relaxin-3 antagonist (R3 B1-22R) based on a modified and truncated relaxin-3 B-chain was recently developed. R3 B1-22R is unstructured in solution; thus, the binding conformation and determinants of receptor binding are unclear. Here, we have designed, chemically synthesized, and pharmacologically characterized more than 60 analogues of R3 B1-22R to develop an extensive understanding of its structure-activity relationships. We show that the key driver for affinity is the nonnative C-terminal Arg23 Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg12, Ile15, and Ile19 Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe14 and Ala17, also interact with RXFP3. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding. These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor.

Highlights

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking
These included Arg8, Arg12, Ile15, Arg16, and Ile19, which all have been shown to be important for relaxin-3 binding [18], amino acid residues that are not surfacedexposed in relaxin-3 due to the interaction with the A-chain, such as Phe14 and Val18 appear to contribute to binding of the antagonist
We have shown here that the antagonist R3 B1-22R likely adopts a helical conformation similar to relaxin-3 upon binding to RXFP3

Summary

Introduction

The neuropeptide relaxin-3 and its receptor relaxin family peptide receptor-3 (RXFP3) play key roles in modulating behavior such as memory and learning, food intake, and reward seeking. We show that R3 B1-22R has a propensity to form a helical structure, and modifications that support a helical conformation are functionally well-tolerated, whereas helix breakers such as proline residues disrupt binding These data suggest that the peptide adopts a helical conformation, like relaxin-3, upon binding to RXFP3, but that its smaller size allows it to penetrate deeper into the orthosteric binding site, creating more extensive contacts with the receptor. Projection of relaxin-3 fibers to the paraventricular nucleus of hypothalamus and the bed nucleus strial terminalis, both regions with high RXFP3 expression, are thought to drive increased food intake and modulate pathways involved in addiction/reward seeking, respectively [3, 6]. Relaxin-3 is, in addition to its cognate receptor, RXFP3, able to activate both RXFP1 and RXFP4 [13,14,15]

Results

Discussion

Conclusion