The electrostatic interactions of relaxin‐3 with receptor RXFP4 and the influence of its B‐chain C‐terminal conformation

• Published in: The FEBS journal 2014-07, Vol.281 (13), p.2927-2936
• Main Authors: Wang, Xin‐Yi, Guo, Yu‐Qi, Zhang, Wei‐Jie, Shao, Xiao‐Xia, Liu, Ya‐Li, Xu, Zeng‐Guang, Guo, Zhan‐Yun
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Amino Acid Substitution; Binding, Competitive; Biophysics; Conserved Sequence; electrostatic interaction; Genes, Reporter; Green Fluorescent Proteins - biosynthesis; Green Fluorescent Proteins - genetics; HEK293 Cells; Hormones; Humans; Insulin; Molecular biology; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Peptides; Protein Binding; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Receptors, G-Protein-Coupled - chemistry; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Receptors, Peptide - chemistry; Receptors, Peptide - genetics; Receptors, Peptide - metabolism; Relaxin - chemistry; Relaxin - genetics; Relaxin - metabolism; relaxin‐3; relaxin‐family peptide; RXFP4
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12830

Relaxin‐3 (also known as insulin‐like peptide 7) is an insulin/relaxin‐superfamily peptide hormone that can bind and activate three relaxin‐family peptide receptors: RXFP3, RXFP4, and RXFP1. Recently, we identified key electrostatic interactions between relaxin‐3 and its cognate receptor RXFP3 by using a charge‐exchange mutagenesis approach. In the present study, the electrostatic interactions between relaxin‐3 and RXFP4 were investigated with the same approach. Mutagenesis of the negatively charged extracellular residues of human RXFP4 identified a conserved EXXXD(100–104) motif that is essential for RXFP4 activation by relaxin‐3. Mutagenesis of the conserved positively charged Arg residues of relaxin‐3 demonstrated that B12Arg, B16Arg and B26Arg were all involved in the binding and activation of RXFP4, especially B26Arg. The activity complementation between the mutant ligands and the mutant receptors suggested two probable electrostatic interaction pairs: Glu100 of RXFP4 versus B26Arg of relaxin‐3, and Asp104 of RXFP4 versus both B12Arg and B16Arg of relaxin‐3. For interaction with the essential EXXXD motifs of both RXFP3 and RXFP4, a folding‐back conformation of the relaxin‐3 B‐chain C‐terminus seems to be critical, because it brings B26Arg sufficiently close to B12Arg and B16Arg. To test this hypothesis, we replaced the conserved B23Gly‐B24Gly dipeptide of relaxin‐3 with an Ala‐Ser dipeptide that occupied the corresponding position of insulin‐like peptide 5 and resulted in an extended helical conformation. The mutant relaxin‐3 showed a significant decrease in receptor‐activation potency towards both RXFP3 and RXFP4, suggesting that a folding‐back conformation of the B‐chain C‐terminus was important for relaxin‐3 to efficiently interact with the EXXXD motifs of both receptors. Structured digital RXFP4 and relaxin-3 physically interact by competition binding (1, 2, 3, 4, 5, 6, 7, 8, 9) Relaxin‐3 is an insulin/relaxin superfamily peptide hormone that can bind and activate three relaxin‐family peptide receptors. In the present work, we disclosed key electrostatic interactions between relaxin‐3 and the receptor RXFP4 through a charge‐exchange mutagenesis approach: Glu100 of RXFP4 interacted with B‐chain C‐terminal B26Arg of relaxin‐3, meanwhile Asp104 of RXFP4 interacted with B‐chain central B12Arg and B16Arg of relaxin‐3.