Solution Structure and Novel Insights into the Determinants of the Receptor Specificity of Human Relaxin-3

Abstract

Relaxin-3 is the most recently discovered member of the relaxin family of peptide hormones. In contrast to relaxin-1 and -2, whose main functions are associated with pregnancy, relaxin-3 is involved in neuropeptide signaling in the brain. Here, we report the solution structure of human relaxin-3, the first structure of a relaxin family member to be solved by NMR methods. Overall, relaxin-3 adopts an insulin-like fold, but the structure differs crucially from the crystal structure of human relaxin-2 near the B-chain terminus. In particular, the B-chain C terminus folds back, allowing Trp B27 to interact with the hydrophobic core. This interaction partly blocks the conserved RXXXRXXI motif identified as a determinant for the interaction with the relaxin receptor LGR7 and may account for the lower affinity of relaxin-3 relative to relaxin for this receptor. This structural feature is likely important for the activation of its endogenous receptor, GPCR135.Recent developments have caused considerable excitement in the relaxin field, including the discovery of a new member of the relaxin family, relaxin-3 (1), and the identification of several long sought after relaxin receptors (2). Prior to the discovery of the relaxin-3 gene (RLN3), only one relaxin gene had been characterized in most mammals, with the exception of humans and higher primates, in which two separate genes, RLN1 and RLN2, were known (3, 4). The product of the human RLN2 gene, human relaxin-2 (H2 relaxin), is the functional ortholog of the RLN1 gene product from non-primate species, which is termed relaxin. The function of the product of the human RLN1 gene, human relaxin-1 (H1 relaxin), is unknown; and indeed, a native H1 relaxin peptide has not been isolated (5). Hence, throughout this work, "relaxin" will refer to the pregnancy hormones H2 relaxin and non-primate relaxin. Relaxin-3 is the ancestor of the entire relaxin peptide family (5), and RLN3 genes have been identified in all mammalian genomes as well as in the genomes of chicken, frog, and various fish species. In contrast, the RLN1 gene is found only in mammals. Fig. 1 shows a sequence comparison of the product of the human RLN3 gene, human relaxin-3 (H3 relaxin); H2 and H1 relaxins; and relaxin-3 orthologs from other species. Interestingly, the relaxin-3 sequences are highly conserved between species, in contrast to relaxin, which shows considerable sequence variation.Relaxin has long been regarded as a hormone mainly associated with pregnancy. Produced in the corpus luteum and/or placenta of most mammals, it has numerous pregnancy-specific actions, including the remodeling of the reproductive tract and preparation of the mammary apparatus for lactation (6). However, relaxin also has important physiological roles outside of pregnancy. It inhibits collagen biosynthesis and promotes collagen breakdown (7-9) and causes vasodilatation in various tissues (10). Interestingly, the expression pattern of relaxin-3 differs significantly from that of relaxin, suggesting a distinctly different physiolog...

Solution Structure and Characterization of the LGR8 Receptor Binding Surface of Insulin-like Peptide 3

“…We recently reported similar broadening for certain resonances in H3 relaxin (18). Interestingly, the same residues are broadened in both peptides, although to a more severe degree in INSL3, with several amide protons being undetectable.…”

“…However the tail following the helix, including the conserved Trp, is flexible and lacks significant electron density in the crystal structure. In contrast, in H3 relaxin we observed a large number of NOEs between the Trp side chain and resonances in the molecular core, including B18, B19, and the A24 -B22 disulfide bond (18). These NOEs were enough to define a conformation in which the tail turns around and the Trp side chain packs against the core and forms favorable hydrophobic interactions.…”

“…4 shows a comparison of the structures of INSL3, H3 relaxin (18), and H2 relaxin (23) from which it is clear that the peptides have a very similar core, with the key differences being mainly around the termini. In both H2 and H3 relaxin the A-chains are highly structured.…”

“…NMR Spectroscopy-Pulsed-field gradient NMR diffusion experiments were performed with a two-dimensional sequence using stimulated echo longitudinal encode-decode (17) as described previously (18). Dioxane (0.2 mM) was used as the internal standard (19) .…”

“…Hydrogen bonds were included into the structure calculations for all amide protons concluded to be slow exchanging once a suitable acceptor could be identified in the preliminary structures. Three-dimensional structures were calculated using simulated annealing and energy minimization protocols from ARIA (20) in the program CNS (21) as described previously (18). Briefly the protocol involves a high temperature phase comprising 4000 steps of 0.015 ps of torsion angle dynamics, a cooling phase with 4000 steps of 0.015 ps of torsion angle dynamics during which the temperature is lowered to 0 K, and finally an energy minimization phase comprising 5000 steps of Powell minimization.…”