Identification of key residues essential for the structural fold and receptor selectivity within the A-chain of H2 relaxin.

H2 relaxin is currently in Phase III clinical trials for the treatment of acute heart failure. It is a 53 amino acid insulin-like peptide comprising of two chains and three disulfide bonds. It interacts with two of the relaxin family peptide (RXFP) receptors. While its cognate receptor is RXFP1, it is also able to cross-react with RXFP2, the native receptor for a related peptide INSL3. In order to understand the basis of this cross-reactivity, it is important to elucidate both binding and activation mechanisms of this peptide. The primary binding mechanism of this hormone has been extensively studied and well defined. H2 relaxin binds to the leucine-rich repeats (LRR) of RXFP1 and RXFP2 using B-chain specific residues. However, little is known about the secondary interaction that involves the A-chain of H2 relaxin and transmembrane exoloops of the receptors. We demonstrate here through extensive mutation of the A-chain that the secondary interaction between H2 relaxin and RXFP1 is not driven by any single amino acid, although residues Tyr3, Leu20 and Phe23 appear to contribute. Interestingly, these same three residues are important drivers of the affinity and activity of H2 relaxin for RXFP2 with additional minor contributions from Lys9, His12, Lys17, Arg18 and Arg22. Our results provide new insights into the mechanism of secondary, activation interaction of RXFP1 and RXFP2 by H2 relaxin, leading to a potent and RXFP1-selective analogue, H2:A(4-24)(F23A), which was tested in vitro and in vivo and found to significantly inhibit collagen deposition similar to native H2 relaxin.