title: The electrostatic interactions of relaxin-3 with receptor RXFP4 and the influence of its B-chain C-terminal conformation.
abstract: 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.
authors.affiliation: Institute of Protein Research, College of Life Sciences and Technology, Tongji University, Shanghai, China; Central Laboratory, Shanghai East Hospital, Tongji University School of Medicine, China.
authors.forename: Xin-Yi
authors.initials: XY
authors.lastname: Wang
authors.forename: Yu-Qi
authors.initials: YQ
authors.lastname: Guo
authors.forename: Wei-Jie
authors.initials: WJ
authors.lastname: Zhang
authors.forename: Xiao-Xia
authors.initials: XX
authors.lastname: Shao
authors.forename: Ya-Li
authors.initials: YL
authors.lastname: Liu
authors.forename: Zeng-Guang
authors.initials: ZG
authors.lastname: Xu
authors.forename: Zhan-Yun
authors.initials: ZY
authors.lastname: Guo
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001667:Binding, Competitive; D017124:Conserved Sequence; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018000:Receptors, Peptide / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012065:Relaxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C480238:RLN3 protein, human; C410092:RXFP3 protein, human; C480239:RXFP4 protein, human; D043562:Receptors, G-Protein-Coupled; D018000:Receptors, Peptide; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012065:Relaxin
keywords: RXFP4; electrostatic interaction; mutagenesis; relaxin-3; relaxin-family peptide