Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids

doi:10.1016/j.biochi.2018.08.003

Biochimie

Volume 154, November 2018, Pages 77-85

https://doi.org/10.1016/j.biochi.2018.08.003 Get rights and content

Highlights

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Receptor selectivity of R3/I5 can be modulated by replacement of B23Gly or B24Gly.
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Unnatural amino acids were incorporated by a sortase-catalysed ligation approach.
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Configuration of the B23 residue affected activation of RXFP3 but not RXFP4.
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Both size and configuration of the B24 residue affected R3/I5 receptor selectivity.
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This study provided new insights into the mechanism of R3/I5 receptor selectivity.

Abstract

Relaxin family peptides perform a variety of biological functions by activating four G protein-coupled receptors, namely RXFP1−4. Our recent study demonstrated that selectivity of the chimeric relaxin family peptide R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of the highly conserved nonchiral B23Gly or B24Gly with some natural l-amino acids. To investigate the mechanism of this modulating effect, in the present study we incorporated unnatural amino acids into the B23 or B24 position of a semi-synthetic R3/I5 that was prepared by a novel sortase-catalysed ligation approach using synthetic relaxin-3 B-chain and recombinant INSL5 A-chain. R3/I5 was a weak agonist for RXFP3 after B23Gly was replaced by D-Ala or D-Ser, but a strong antagonist for this receptor after B23Gly was replaced by corresponding l-amino acids. However, these replacements always resulted in a weak agonist for RXFP4. Thus, configuration of the B23 residue of R3/I5 affected activation of RXFP3 but not RXFP4. For the B24 residue, both size and configuration affected receptor selectivity of R3/I5. l-amino acids with an appropriate size, such as L-Ser and L-Abu, had the greatest effect on increasing the selectivity of R3/I5 towards RXFP3 over the homologous RXFP4. Our present results provided new insights into receptor selectivity of R3/I5, and would facilitate design of novel agonists or antagonists for RXFP3 and RXFP4 in future studies.

Introduction

The relaxin family, a branch of the insulin superfamily, is a group of homologous peptide hormones that includes relaxin, relaxin-3, and insulin-like peptide 3–6 (INSL3−6). These peptides perform a variety of biological functions, such as regulating reproduction, food intake, stress responses, and glucose homeostasis, by activating four A-class G protein-coupled receptors, namely RXFP1−4 [[1], [2], [3], [4], [5]]. Relaxin and INSL3 are the cognate agonists of the homologous RXFP1 and RXFP2, respectively [6,7], while relaxin-3 and INSL5 are the cognate agonists of the homologous RXFP3 and RXFP4, respectively [8,9]. In addition, relaxin-3 can also activate RXFP1 and RXFP4 in vitro with high efficiency [10,11]. The receptors of INSL4 and INSL6 remain unknown.

In the relaxin family, relaxin-3 and INSL5 share the highest sequence homology, but they display distinct receptor selectivity: relaxin-3 can activate RXFP3, RXFP4, and RXFP1 with high efficiency, while INSL5 can only activate RXFP4. A previously designed chimeric R3/I5 peptide, containing the B-chain of relaxin-3 and the A-chain of INSL5, retains high activation potency towards both RXFP3 and RXFP4, but loses activity towards RXFP1 [12], suggesting that the A-chain of INSL5 and relaxin-3 determines their different selectivity against RXFP1. As shown in Fig. 1, mature R3/I5 is comprised of two polypeptide chains with three disulfide linkages and folds into a globular structure similar to that of relaxin-3 [13,14]. Both have a folded-back conformation at the B-chain C-terminus, probably due to high flexibility of the smallest nonchiral Gly residue at their B23 and B24 positions. By contrast, the B-chain C-terminus of INSL5 adopts an extended α-helical conformation [15], likely because its corresponding positions (B20 and B21) are occupied by larger residues, typically Ala and Ser, respectively. Our recent studies revealed that selectivity of relaxin-3, INSL5, and R3/I5 towards the homologous RXFP3 and RXFP4 can be modulated by replacement of their residues responsible for the B-chain C-terminal conformation with some natural amino acids [[16], [17], [18], [19]]. For examples, replacement of the highly conserved nonchiral B23Gly with L-Ala or L-Ser converted R3/I5 from an efficient agonist to a strong antagonist for RXFP3, but the mutants retained weak activation potency towards RXFP4; replacement of the highly conserved nonchiral B24Gly with some l-amino acids increased selectivity of R3/I5 towards RXFP3 over the homologous RXFP4, and resulted in the best selective agonist [Gly(B24)L-Ser]R3/I5 for RXFP3 [19]. However, the mechanism for this modulating effect is not yet understood. To investigate the mechanism, in the present work we attempted to incorporate some unnatural amino acids into the B23 or B24 position and test their effects on receptor selectivity of R3/I5.

To incorporate unnatural amino acids into the B23 or B24 position of R3/I5, chemical synthesis of the mutant B-chains offers a practical approach. However, combining the separate A-chain and B-chain is difficult and this hampers wide application of the chemical synthesis approach in most biological laboratories. In recent years, our laboratory designed single-chain precursors for relaxin family peptides that can be efficiently overexpressed in Escherichia coli, spontaneously refolded in vitro, and then enzymatically converted to mature two-chain forms under mild conditions [[20], [21], [22], [23], [24], [25]]. In the present work, we developed a sortase-catalysed ligation approach using synthetic relaxin-3 B-chain harbouring a sortase recognition motif at the C-terminus and a recombinant INSL5 A-chain harbouring four successive Gly residues at the N-terminus (Fig. 2A). Once the synthetic B-chain and recombinant A-chain were ligated together, the single-chain R3/I5 precursor could spontaneously form the correct disulfide linkages in vitro with high efficiency, and be converted to the mature form by enzymatic treatment under mild conditions. This sortase-catalysed ligation approach does not require any special modification of the synthetic wild-type or mutant B-chains, hence they are readily available for most laboratories via custom peptide synthesis. Sortases are a group of bacteria-derived enzymes catalysing transpeptidation for anchoring some surface proteins to the cell wall [[26], [27], [28]], and have been used for protein/peptide modification in recent years [[29], [30], [31], [32]]. Using this sortase-catalysed ligation approach, in the present study we prepared six mutant R3/I5s bearing unnatural amino acids at the B23 or B24 position, and investigated their selectivity towards the homologous RXFP3 and RXFP4.

Section snippets

Preparation of synthetic relaxin-3 B-chain and recombinant INSL5 A-chain

Ligation versions of wild-type or mutant relaxin-3 B-chain, harbouring a sortase recognition motif at the C-terminus, were chemically synthesised via solid-phase peptide synthesis using standard Fmoc methodology (GL Biotech, Shanghai, China). Crude peptides were purified to ∼90% homogeneity by high performance liquid chromatography (HPLC) using a C18 reversed-phase column (Zorbax 300SB single bond C18, 9.4 × 250 mm; Agilent Technologies, Santa Clara, CA, USA). The ligation version of recombinant INSL5

Development of a sortase-catalysed ligation approach for preparation of semi-synthetic R3/I5

To develop the sortase-catalysed ligation approach, we introduced a sortase recognition motif to the C-terminus of the synthetic relaxin-3 B-chain and tested several versions (supplementary Table S1). All B-chain versions were successfully synthesised by standard solid-phase peptide synthesis, but only the version shown in Fig. 2A displayed high solubility (∼1 mM) in ligation buffer, and exhibited high ligation efficiency (∼70%) with recombinant INSL5 A-chain. A previous study demonstrated that

Discussion

In the present work, we developed a novel sortase-catalysed ligation approach for preparation of the chimeric R3/I5 peptide using synthetic B-chain and recombinant A-chain. Through this approach, various R3/I5 mutants bearing unnatural amino acids in the B-chain could be quickly prepared. Wild-type or mutant B-chains are readily available from custom peptide synthesis, and ligation, refolding, and maturation are conducted under mild conditions, hence the sortase-catalysed ligation approach can

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (31670773, 31470767).

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Citation Excerpt :
Meanwhile, the large aromatic Trp residue probably forms a hydrophobic interaction with the B-chain C-terminal Trp residue of the agonists. Novel agonists and antagonists have been designed for RXFP3 in previous studies [24–32], such as the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 (Fig. 1B). The chimeric agonist R3/I5 contains the B-chain of relaxin-3 and the A-chain of INSL5.
Relaxin family peptide receptor 3 (RXFP3) is a G protein-coupled receptor implicated in the regulation of food intake and stress response upon activation by the neuropeptide relaxin-3. In recent studies, interactions of RXFP3 with some natural or synthetic ligands have been investigated. In the present study, we identified the hydrophobic interactions of human RXFP3 with the chimeric agonist R3/I5 and the chimeric antagonist R3(ΔB23-27)R/I5 using a newly developed NanoBiT-based homogenous binding assay. We first demonstrated that the conserved large aliphatic B15Ile and B19Ile were important for the binding of the agonist and antagonist to RXFP3, because alanine replacement significantly decreased their receptor-binding potency. Thereafter, we demonstrated that the conserved large aliphatic Leu246 and Leu248 in extracellular loop 2 were important for RXFP3 binding to the agonist and antagonist, because alanine replacement significantly decreased the binding affinity of RXFP3 for both ligands. Finally, we deduced probable hydrophobic interactions based on the ability of RXFP3 mutants to distinguish the wild-type and mutant ligands: Leu246 of RXFP3 interacted with B15Ile of both ligands, while Leu248 of RXFP3 interacted with both B15Ile and B19Ile of the agonist and antagonist. The present results not only provided new insights into the interaction mechanism of RXFP3 with agonists and antagonists, but also demonstrated usefulness of the NanoBiT-based homogenous binding assay to study the interaction mechanism of certain receptors with their ligands.
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¹: These authors contributed equally to this work.

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Research paperExploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids

Highlights

Abstract

Introduction

Section snippets

Preparation of synthetic relaxin-3 B-chain and recombinant INSL5 A-chain

Development of a sortase-catalysed ligation approach for preparation of semi-synthetic R3/I5

Discussion

Acknowledgments

Curr. Opin. Chem. Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

Peptides

Process Biochem.

Biochim. Biophys. Acta

Curr. Opin. Struct. Biol.

Curr. Opin. Chem. Biol.

International Union of Basic and Clinical Pharmacology. XCV. Recent advances in the understanding of the pharmacology and biological roles of relaxin family peptide receptors 1-4, the receptors for relaxin family peptides

Pharmacol. Rev.

Relaxin family peptides and their receptors

Physiol. Rev.

Relaxin-like peptides in male reproduction - a human perspective

Br. J. Pharmacol.

Research paper
Exploring receptor selectivity of the chimeric relaxin family peptide R3/I5 by incorporating unnatural amino acids