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...