Abstract
The relaxin-3 neuropeptide activates the relaxin family peptide 3 (RXFP3) receptor to modulate stress, appetite, and cognition. RXFP3 shows promise as a target for treating neurological disorders, but realization of its clinical potential requires development of smaller RXFP3-specific drugs that can penetrate the blood-brain barrier. Designing such drugs is challenging and requires structural knowledge of agonist- and antagonist-binding modes. Here, we used structure-activity data for relaxin-3 and a peptide RXFP3 antagonist termed R3 B1-22R to guide receptor mutagenesis and develop models of their binding modes. RXFP3 residues were alanine-substituted individually and in combination and tested in cell-based binding and functional assays to refine models of agonist and antagonist binding to active- and inactive-state homology models of RXFP3, respectively. These models suggested that both agonists and antagonists interact with RXFP3 via similar residues in their B-chain central helix. The models further suggested that the B-chain Trp27 inserts into the binding pocket of RXFP3 and interacts with Trp138 and Lys271, the latter through a salt bridge with the C-terminal carboxyl group of Trp27 in relaxin-3. R3 B1-22R, which does not contain Trp27, used a non-native Arg23 residue to form cation-π and salt-bridge interactions with Trp138 and Glu141 in RXFP3, explaining a key contribution of Arg23 to affinity. Overall, relaxin-3 and R3 B1-22R appear to share similar binding residues but may differ in binding modes, leading to active and inactive RXFP3 conformational states, respectively. These mechanistic insights may assist structure-based drug design of smaller relaxin-3 mimetics to manage neurological disorders.
Highlights
RXFP3 activation; we propose that it is interacting with relaxin-3 PheB20 (Fig. 5B)
Given the involvement of Glu[244] and Asp[145], we predicted that the hydrophobic patch on EL2 of RXFP3 could retain interactions with Ile[15] and Ile[19] of R3 B1–22R, as for relaxin-3
H268A, T346A, and S349A resulted in a decrease in antagonist affinity (ϳ2–3-fold) but not agonist binding or potency, suggesting that these amino acid residues are interacting only with the antagonist (Fig. 6C, Fig. S2, and Table 1)
Summary
In previous studies exploring the role of acidic amino acid residues in RXFP3 for relaxin-3 binding, interaction partners for the key basic amino acid residues in relaxin-3, ArgB12, ArgB16, and ArgB26, were identified (34, 35) (Fig. 2A). Eight of 15 mutations tested (W138A (TM2); T162A (TM3); V241A, L246A, and L248A (EL2); K271A (TM5); and F364A and F371A (TM7)) resulted in a significant loss (ϳ70 – 85% compared with WT) in Eu-R3/I5–specific binding without affecting receptor cell surface expression (Fig. 3 and Table 1). When the mutant receptors were tested in cAMP activity assays, 9 of 15 mutations demonstrated significantly reduced agonist potency (pEC50) compared with WT RXFP3 (Fig. 4 and Table 1). In our previous study (34), we generated a homology model of RXFP3 and performed ligand-docking simulations guided by our mutagenesis data based on the inactive structure of the CXCR4 receptor (PDB entry 3OEO (43)) We have utilized this model to predict further interaction sites and perform more RXFP3 alanine mutations in this study.
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