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

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 Arg Additional contributors to binding include amino acid residues that are important also for relaxin-3 binding, including Arg, Ile, and Ile Intriguingly, amino acid residues that are not exposed in native relaxin-3, including Phe and Ala, 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.

Development of Relaxin-3 Agonists and Antagonists Based on Grafted Disulfide-Stabilized Scaffolds

“…The NMR structural analysis confirmed that the apamin scaffold does confer helical structure, but that the C-terminal part of the peptide after the last cysteine, which includes the key antagonist binding residue ArgB23, remains highly flexible. Again this is consistent with detailed structure activity analysis of R3 B1-22R, which suggest a degree of flexibility is a prerequisite for optimal binding (Haugaard-Kedström et al, 2018;Wong et al, 2018). The VhTI grafted antagonist was based on the first version of the VhTI agonist and retained the native Pro residue from VhTI instead of ArgB12.…”

“…The VhTI grafted antagonist was based on the first version of the VhTI agonist and retained the native Pro residue from VhTI instead of ArgB12. The relative importance of ArgB12 for binding to RXFP3 in antagonists is significantly less than in native relaxin-3 (Haugaard-Kedström et al, 2018;Wong et al, 2018), thus we did not anticipate this being detrimental to affinity. However, analog 10 showed very poor binding to RXFP3 (Table 2, Figure 6), which again is likely related to the positioning of the binding residues on the helical surface.…”

“…In order to further support the helical propensity of analog 3, analog 4 also includes aminoisobutyric acid (Aib) amino acids, known to induce helicity due to the stereochemical constraint of an additional methyl group at the Cα carbon (Mahalakshmi and Balaram, 2006). Val18 and Thr21 of relaxin-3 B-chain were substituted with Aib in analog 4, as those residues are not important for activity of relaxin-3 (Kuei et al, 2007), and a recent study has shown that Aib can be incorporated at these positions without adversely affecting binding of R3 B1-22R to RXFP3 (Haugaard-Kedström et al, 2018). Analog 5 is based on the VhTI scaffold and incorporates residues equivalent to ArgB8, IleB15, ArgB16, PheB20, and the C-terminal tail.…”

“…Furthermore, structural analysis using NMR showed the peptide was largely unstructured rather than being able to retain the VhTI fold, which would explain the lack of potency. We have previously shown that the single chain antagonist R3 B1-22R is able to bind efficiently to RXFP3 without being structured in solution (Haugaard-Kedström et al, 2011), and in fact the flexibility is favored for being able to adapt to bind efficiently to RXFP3 in a slightly different conformation relative to native relaxin-3 (Haugaard-Kedström et al, 2018;Wong et al, 2018).…”

“…Important relaxin-3 B-chain residues involved in binding and activation of RXFP3 are shown in sticks and colored blue and green for basic and hydrophobic residues, respectively. structure (Haugaard-Kedström et al, 2018;Wong et al, 2018). For single chain agonists the affinity can instead be improved by reintroducing helical structure in the absence of the A-chain through "helical stapling" using dicarba bonds (Hojo et al, 2016;Jayakody et al, 2016).…”